Human dorsal tissue affecting factor (noggin) and nucleic acids encoding same

ABSTRACT

Novel dorsal growth inducing factors, complexes including the factors, and DNA or RNA coding sequences for the factors are described.

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/957,401 filed on Oct. 6, 1992, now abandoned, which has acontinuation-in-part of U.S. patent application Ser. No. 07/950,410filed on Sep. 23, 1992, now abandoned, which was a continuation-in-partof U.S. patent application Ser. No. 07/939,954 filed on Sep. 3, 1992,now abandoned.

FIELD OF THE INVENTION

The invention generally relates to growth factors and neurotrophicfactors, and more particularly to a soluble growth factor with dorsalgrowth inducing activity, to complexes including the factor, and to DNAor RNA coding sequences for the factor.

This invention was made, in part, with government support under GrantContract No. ROI-GM-42341, awarded by the National Institutes of Health.The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Growth factors are substances, such as polypeptide hormones, whichaffect the growth of defined populations of animal cells in vivo or invitro, but which are not nutrient substances. Proteins involved in thegrowth and differentiation of tissues may promote or inhibit growth, andpromote or inhibit differentiation, and thus the general term "growthfactor" includes cytokines and trophic factors. Among growth, orneurotrophic factors presently known are those that can be classifiedinto the insulin family insulin, insulin-like growth factors (e.g.,IGF-I, IGF-II), mammary stimulating factor (MSF), and nerve growthfactor (NGF)!; those classified into the epidermal growth factor familyepidermal growth factor (EGF) and transforming growth factors (TGFα,TGFβ, TGFγ)!; those classified into the platelet-derived growth factorfamily platelet-derived growth factor (PDGF), osteosarcoma-derivedgrowth factor (ODGF), and fibroblast growth factor (FGF)!; theneurotrophins nerve growth factor (NGF), brain derived neurotrophicfactors (BDNF) neurotrophins 3, 4, 5, (NT-3, NT-4, NT-5)!; and otherscolony stimulating factor (CSF), T-cell growth factor, tumorangiogenesis factor (TAF), DNA synthesis promoting factor (DSF),tumor-derived growth factors, fibroblast-derived growth factor (FDGF)!.

Receptors that affect growth (that is, receptors for growth-associatedligands) are proteins found associated with cell surfaces thatspecifically bind their growth factors as ligands. Growth factorreceptors are utilized in various clinical and diagnostic applications.

U.S. Pat. No. 4,857,637, issued Aug. 15, 1989, inventors Hammonds etal., describes a method for immunizing an animal against its growthhormone receptor through use of vaccinating with antibodies in order tostimulate growth of the animals.

U.S. Pat. No. 4,933,294, issued Jun. 12, 1990, inventors Waterfield etal., describes studies of structural alterations of the human EGFreceptor and its gene and a relationship in tumorigenesis for assays andtherapies involving the human EGF receptor. For example, such assays caninvolve detection of structurally altered or abnormally expressed growthfactor receptor and the mRNA transcripts and genes which encode them.EGF may have a role in cell proliferation and differentiation since itinduces early eyelid opening and incisor development in new born mice.

U.S. Pat. No. 5,030,576, issued Jul. 9, 1991, inventors Dull et al.,describes the role of receptors, such as receptors for growth factors,in designing drugs by the pharmaceutical industry, and discloses use ofa receptor hybrid for screening drug purposes, such as in studies of EGFbinding domains. U.S. Pat. No. 5,087,616, issued Feb. 11, 1992,inventors Myers and Bichon, describes a method for destroying tumorcells with a composition including a drug conjugate. The conjugate has agrowth factor as one moiety and a polymeric carrier with a cytotoxiccompound as another moiety. Thus, compositions of the patent aredescribed as binding preferentially to tumor cells bearing EGF-bindingreceptors (when an EGF growth factor, for example, is used as a firstmoiety).

U.S. Pat. No. 5,098,833, issued Mar. 24, 1992, inventors Lasky, et al.,describes a DNA isolant capable of hybridizing to the epidermal growthfactor domain. Expression systems for recombinant production are said tobe useful in therapeutic or diagnostic compositions.

A good background review of a neurotrophic factor related to NGF isprovided by W092/05254, published Apr. 2, 1992, which also describesstate of the art methods of: preparing amino acid sequence variations,site-directed mutagenesis techniques, ligation of coding DNA into areplicable vector for further cloning or for expression, choice ofpromoters for expression vectors, suitable host cells for expression,particularly mammalian cells, protein purification upon recovery fromculture medium as a secreted protein, derivatization with bifunctionalagents to cross-link protein to a support matrix for use withantibodies, entrapment in systems for drug delivery, preparation oftherapeutic formulations, and methods of administration. In addition,preparation of polyclonal and monoclonal antibodies are described, suchas are useful in diagnostic assays. These various aspects of isolation,preparation, and applications for a novel neurotrophic factor, asillustrated by the W092/05254 publication, are incorporated herein byreference.

Thus, growth factors, their receptors, and DNA or RNA coding sequences,therefore, and fragments thereof are useful in a number of therapeutic,clinical, research, diagnostic, and drug design applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequence (SEQ ID NO:1) for the human noggin gene anddeduced amino acid sequence (SEQ ID NO:2).

FIG. 2 (A). Experimental design: competent animal cap(AC) ectoderm wasdissected from staged embryos as shown. St10.5 dorsal and ventral AC andventral marginal zones (VMXZ) also dissected as shown. Explants werewashed once in low Ca/Mg Ringers (LCMR) solution and then placed intreatment medium containing factor diluted in LCMR+0.5% BSA. Explantscultured to late stages (St20+) were removed from treatment medium 6-16hours after the start of treatment and placed in LCMR. When explantsreached the desired stage they were either harvested for RNA, or theywere fixed for whole mount in situ hybridization or antibody staining.

FIG. 2 (B). Neural induction by noggin in the absence of muscle. Lanes1-3 show specific fragments protected by N-CAM, β-tubulin, and XIF-3probes respectively in whole St24 embryo RNA. Lanes 4-8 show protectionby the mixture of these three probes while lanes 9-13 show protection byan actin probe on tRNA(t), St24 embryo RNA (E), and RNA collected fromSt9 AC treated with 50pM activin (A), 25% of 20 fold concentratedcontrol CHO cell medium (C) or 25% of 20 fold concentrated nogginconditioned CHO cell medium (N). Ubiquitously expressed cytoskeletalactin used as a loading control shows that RNA levels in all treatmentsare comparable (lanes 11-13).

FIG. 3. 12% SDS-PAGE run under reducing conditions. Proteins werevisualized by silver staining. Lane 1 shows molecular size standards.Lanes 2-7 show 0, 0.1, 0.2, 0.5, 1, and 2 μg of purified human noggin.

FIG. 4 (A). Time course of animal caps treated with purified noggin vs.activin; direct vs indirect neural induction. Animal caps were dissectedas shown in FIG. 2A and treated with LCMR+0.5% BSA (U), a 20% dilutionof activin conditioned COS cell medium (A), or 1 μg/ml purified humannoggin(N). RNA isolated from treated animal caps (lanes 2-13) along withSt22 whole embryo RNA (lane 1) and tRNA (lane 14) was probed for N-CAM,β-tubulin, muscle and cytoskeletal actins, collagen type II, and EF-1a.

FIG. 4 (B). Expression of early mesoderm markers in activin but notnoggin induced animal caps. Animal caps were dissected from St8 embryos,treated as described in (A), and harvested at St11. Lanes 1 and 2respectively show goosecoid and Xbra probe protection by St10.5 wholeembryo RNA. Lanes 3-6 show protection by a mix of these two probes.Relative RNA levels are demonstrated by separate EF-1α probe protection.

FIG. 4 (C). Plasmid directed gastrula stage noggin expression directlyinduces neural tissue. One cell stage embryos were injected with 20 pgof pCSKAlacZ or pCSKAnoggin into the animal pole. Animal caps frominjected embryos were dissected at St8-9 and cultured until St20, whenthey were harvested for analysis by RNase protection.

FIG. 5. Responsiveness of dorsal and ventral animal caps to neuralinduction by noggin. St 105 ventral and dorsal animal caps weredissected as shown in FIG. 2. Dorsal and ventral animal caps weretreated with activin medium (DA, VA) or 1 μg/ml human noggin (DN, VN)and harvested at St26 for RNase protection analysis using N-CAM,β-tubulin, and actin as probes.

FIG. 6. Dose response of ventral marginal zones and animal caps to humannoggin protein. St 10.5 VMZs and St9 animal caps were dissected as shownin FIG. 2A., and treated with 0, 1, 10, 50, 200, and 1000 ug/ml of humannoggin (lanes 3-8 and 10-15 respectively). RNA from treated explants andcontrol whole embryos aged to St26 was then analyzed by RNaseprotection, using the probes N-CAM, β-tubulin, actin and collagen typeII. In this experiment, muscle induction at the dose of 1 ng/ml isstronger than at 10 ng/ml, and there is a low level of muscle actinexpression in the uninduced VMZs. This could be due to experimentalvariability since in repeated experiments we saw muscle induction onlyat the doses of 50 ng/ml and above (data not shown).

FIG. 7 (A-B). In situ hybridization and antibody staining. Tailbudembryos stained for NCAM showing side and dorsal views (a, b); NCAM RNAis only detected in the neural tube, and not the somites. Forcomparison, somites of a tailbud embryo stain for muscle actin, dorsalview (c). Neural specific 6F11 antibody staining at St30 (d-f). Somecement gland pigment remained in these embryos after bleaching as seenin (d), however this pigment is distinct from antibody staining. Theinner mass of staining in the noggin treated animal caps is due to the6F11 antibody detection. Cement gland specific XAG-1 transcriptsdetected at St23 (g-i), and anterior brain otxA transcripts detected atSt35 (j-l) in whole embryos at (d, g, j), human noggin treated (1 μg/ml)animal caps (e, h, k), and untreated animal caps (f, i, l).

FIG. 8. Reverse phase HPLC profile of two refolded isoforms of noggin.The refolded noggin solution was applied onto a Brownlee AquaporeAX-300, 0.46×22 cmHPLC column at a flow rate of 1 ml/min. The column wasequilibrated with solvent A containing 0.1% TFA in water. Solvent B was0.1% TRA in acetonitrile. The column was developed according to thefollowing protocol: a) 2 min isocratically at 95% of solvent A-5% ofsolvent B; 60 min linear gradient to 65% of solvent B and 35% of solventA. Correctly refolded noggin elutes earlier at 44%-46% solvent B.

FIG. 9. Reverse-phase HPLC chromatography characterization ofrecombinant noggin refolded and purified from E. coli. Conditions as inthe legend to FIG. 8.

FIG. 10. Recombinant noggin produced in E. coli and in insect cellsanalyzed by 12.5% SDS-PAGE. Lanes H, L: High and low molecular weightmarkers of the indicated size, respectively. Lanes 1, 2: Recombinantnoggin produced in E. coli and in insect cells respectively, treatedwith 2-mercaptoethanol before electrophoresis. The slower mobility ofnoggin from insect cells corresponds to the size increase that wouldoccur due to N-linked glycosylation at the single consensus site. Lanes2, 3: Recombinant noggin produced in E. coli and in insect cellsrespectively, not treated with 2-mercaptoethanol before electrophoresis.

FIG. 11. Circular dichroism spectra of recombinant noggin produced in E.coli (--), and in insect cells (-).

FIG. 12. Ventral marginal zone assay showing induction of muscle actinmRNA after exposure to human noggin (0.01, 0.05, 0.2 μg/ml) produced inbaculovirus, a mock transfected culture of baculovirus (0.02, 1 μg/ml)or human noggin produced in E. coli (0.1, 0.5, 2, or 10 μg/ml).

FIG. 13. Nucleotide sequence (SEQ ID NO:25) for the mouse noggin geneand deduced amino acid sequence (SEQ ID NO:26).

SUMMARY OF THE INVENTION

In one aspect of the present invention a peptide that can be insubstantially purified form is characterized by one or more of thefollowing, highly conserved amino acid sequences:

QMWLWSQTFCPVLY (SEQ ID NO:3);

RFWPRYVKVGSC (SEQ ID NO:4);

SKRSCSVPEGMVCK (SEQ ID NO:5);

LRWRCQRR (SEQ ID NO:6); and,

ISECKCSC (SEQ ID NO:7).

Peptides of the invention induce dorsal growth in vertebrates and can beprepared in soluble, physiologically active form for a number oftherapeutic, clinical, and diagnostic applications.

In a preferred embodiment, human noggin protein as set forth in FIG. 1(SEQ ID NO:2) is prepared for use in therapeutic, clinical anddiagnostic applications.

In another aspect of the present invention an oligonucleotide, such ascDNA, is provided having substantial similarity to (or being the sameas) SEQ ID NO:8 (deduced amino acid sequence, SEQ ID NO:9), SEQ ID NO:10(deduced amino acid sequence, SEQ ID NO:11), or SEQ ID NO:1. Thisoligonucleotide can be single or double stranded, be formed of DNA orRNA bases, and can be in the antisense direction with respect to SEQ IDNOS:8, 10 or 1. SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:1 each code fora functional polypeptide that we have designated "noggin," which iscapable of inducing dorsal development in vertebrates when expressed.

Noggin or fragments thereof (which also may be synthesized by in vitromethods) may be fused (by recombinant expression or in vitro covalentmethods) to an immunogenic polypeptide and this, in turn, may be used toimmunize an animal in order to raise antibodies against a nogginepitope. Anti-noggin is recoverable from the serum of immunized animals.Alternatively, monoclonal antibodies may be prepared from cells to theimmunized animal in conventional fashion. Antibodies identified byroutine screening will bind to noggin but will not substantiallycross-react with "wnt" or other growth factors. Immobilized anti-nogginantibodies are useful particularly in the diagnosis (in vitro or invivo) or purification of noggin.

Substitutional, deletional, or insertional mutants of noggin may beprepared by in vitro or recombinant methods and screened forimmuno-crossreactivity with noggin and for noggin antagonist or agonistactivity.

Noggin also may be derivatized in vitro in order to prepare immobilizednoggin and labelled noggin, particularly for purposes of diagnosis ofnoggin or its antibodies, or for affinity purification of nogginantibodies.

The present invention further provides for expression of biologicallyactive noggin molecules in prokaryotic and eukaryotic expressionsystems.

The present invention further provides for the production of noggin inquantities sufficient for therapeutic and diagnostic applications.Likewise, anti-noggin antibodies may be utilized in therapeutic anddiagnostic applications. For most purposes, it is preferable to usenoggin genes or gene products from the same species for therapeutic ordiagnostic purposes, although cross-species utility of noggin may beuseful in specific embodiments of the invention.

In additional embodiments, the noggin nucleic acids, proteins, andpeptides of the invention may be used to induce neural tissue formationin mammals.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have discovered a structurally unique growth factor that is readilyavailable in substantially pure, soluble form. We have named theinventive polypeptide "noggin." This newly isolated neurotrophic factorinduces dorsal development in vertebrates.

An earlier described family of proteins that also induces dorsaldevelopment are the "wnt" proteins. These, however, in contrast tonoggin remain tenaciously bound to cell surfaces. Our initial work withnoggin has been in Xenopus embryos; however, noggin is highly conservedamong vertebrates, as our work with mouse noggin has demonstrated. Theprior known FGF growth factor family is also known to be involved inearly embryonic induction, but both the FGF proteins and their receptorsare distinctly different from noggin. Noggin modifies the actions of FGF(and also activin), for example by potentiating growth, and is thusparticularly suggested in therapeutic compositions for use incombination with other growth factors (as therapeutic adjuvants), suchas to modify or potentiate their effects.

We have cloned cDNA for noggin. The noggin cDNA contains a singlereading frame encoding a 26 kDa protein with a hydrophobicamino-terminal sequence. Noggin is secreted. Noggin's cDNA encodes theprotein as a 26 kDa protein, but we have determined that noggin issecreted in vivo, apparently as a dimeric glycoprotein with a startingapparent molecular weight of about 33 kDa (as the wild-type subunit).When not glycosylated, the monomeric unit has an apparent molecularweight on SDS PAGE of about 25-30 kDa.

We have cloned the gene for human noggin (FIG. 1; SEQ ID NO: 1). Thesequence codes for a protein which has noggin activity (SEQ ID NO:2).The carboxy terminal region of noggin shows homology to a Kunitz-typeprotease inhibitor, indicating that noggin protein, or fragmentsthereof, may exhibit activities of a protease inhibitor.

We have been able to express biologically active noggin in botheucaryotic and prokaryotic host cells. Two expression systems we havesuccessfully used to express biologically active noggin have beenmammalian cell lines (COS and mouse 293). A third expression system isinjection of synthetic mRNA into Xenopus oocytes. In addition, we havesuccessfully expressed biologically active human noggin in a prokaryoticsystem, E. coli, and in baculovirus.

Expression in these several different systems also illustrates the highdegree of conservation for noggin. We have found, for example,substantial sequence similarity between frog noggin and mouse nogginwith a number of completely conserved stretches. Thus, the followingamino acid sequences represent completely conserved portions as betweenfrog noggin and mouse noggin:

QMWLWSQTFCPVLY (SEQ ID NO:3);

RFWPRYVKVGSC (SEQ ID NO:4);

SKRSCSVPEGMVCK (SEQ ID NO:5);

LRWRCQRR (SEQ ID NO:6); and,

ISECKCSC (SEQ ID NO.7).

There is about 87% overall conservation between the mouse and frogsequences, and we have also observed a unique cysteine distributionbetween the two.

Noggin nucleic acids, or oligonucleotides, encode a noggin polypeptideor hybridize to such DNA and remain stably bound to it under stringentconditions and are greater than about 10 bases in length; provided,however, that such hybridizing nucleic acid is novel and unobvious overany prior art nucleic acid including that which encodes or iscomplementary to nucleic acid encoding other growth factors.

By "stringent conditions" we mean are those which (1) employ low ionicstrength and high temperature for washing, for example, 0.15MNaCl/0.015M sodium citrate/0.1% NaDodSo₄ at 50° C., or (2) use duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.

By "substantial similarity," when we are referring to a nucleotidesequence, is meant cross hybridization of sequences under conditions ofmoderate stringency using a probe greater than 100 nucleotides long at30° C. in a standard buffer (Wahl et al., PNAS,76, 3683) and washes at37° C. in 300 mM NaCl, 30 mM sodium citrate, 0.2% SDS at pH 7.Alternatively, one is able to isolate, by polymerase chain reaction, afragment of DNA coding for noggin or noggin family members when usingprimers of degenerate sequence that encode those SEQ ID NOS:3-7.

By "substantial similarity" when reference is made to proteins is thatnoggin from different species, or noggin family members within aspecies, will preserve the positions of cysteine residues in at least80% of positions throughout the protein. Like the neurotrophin family,the sequence of the mature form of noggin and noggin relatedpolypeptides will be identical in at least 40% of positions. Substantialsimilarity at the protein level includes an ability of a subject proteinto compete with noggin for binding to receptors and some (but not all)monoclonal antibodies raised against noggin epitopes.

The cloned cDNA for noggin (derived from frog) is designated herein asSEQ ID NO:8, partial sequence from mouse as SEQ ID NO:10 or fullsequence of mouse noggin as shown in FIG. 13 (SEQ ID NO:25). The humansequence is designated herein as SEQ ID NO:1. We have used RNAtranscripts from the SEQ ID NO:8 clone to rescue embryos and return themto substantially normal development when the noggin RNA is injected intoventralized embryos. In high doses this results in excessive headdevelopment and it is for this reason we named the protein "noggin." Innorthern blot analysis the noggin cDNA hybridizes to two mRNAs that areexpressed both maternally and zygotically.

When using nucleotide sequences coding for part or all of noggin inaccordance with this invention, the length of the sequence should be atleast sufficient in size to be capable of hybridizing with endogenousmRNA for the vertebrate's own noggin. Typically, sufficient sequencesize (for example, for use as diagnostic probes) will be about 15consecutive bases (DNA or RNA). In some diagnostic and therapeuticapplications, one may wish to use nucleotide noggin coding sequences(analogous to all or a portion of SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:25 or SEQ ID NO:1) in the anti-sense direction with respect to eitherSEQ ID NOS:8, 10, 25, or 1.

We suggest as a few preferred primers for amplifying noggin from otherspecies (e.g. human):

5' Primer 1 SEQ ID NO:12

C A A/G A C N T T C/T T G C/T C C N G T N

5' Primer 2 SEQ ID NO:13

T T C/T T G G C C N C/A G N T A C/T G T N A A A/G G T N G G

5' Primer 3 SEQ ID NO:14

C C N G A A/G G G N A T G G T N T G

3' Primer 1 SEQ ID NO:15

C A N C/G T/A A/G C A C/T T T A/G C A C/T T C

3' Primer 2 SEQ ID NO:16

C A N A C C A T N C C C/T T C N G G

3' Primer 3 SEQ ID NO:17

C G/T N C G/T T/C T G G/A C A N C G/T C C A

where N represents a mixture of all four nucleotides and mixtures of twonucleotides are represented by alternates (e.g. A/G).

Although noggin transcript is not localized in the oocyte and cleavagestage embryo, zygotic transcripts are initially restricted to thepresumptive dorsal mesoderm, and reach their highest levels at thegastrula stage in the dorsal lip of the blastopore (Spemann'sorganizer). In the neurula, noggin is transcribed in the notochord andprechordal mesoderm.

Without being bound by theory, we have formulated hypotheses about theembryological effects of noggin based on where it is expressed, and onthe effects of RNA injection in embryos. Since noggin is expressed inthe Spemann organizer, we believe noggin to be a mediator of the effectsof the Spemann organizer, namely neural induction and dorsalization ofthe mesoderm. We have shown that noggin is able to directly induceneural tissue formation. Since noggin is expressed in the notochord andhead mesoderm, we believe noggin to influence either the dorsal-ventralpattern or anterior-posterior pattern of the neural plate. Since nogginis expressed in the branchial arch neural crest, we believe it maytherefore influence whether neural crest cells deposit cartilage andalso to influence later branchial arch growth and remodelling. Noggin isexpressed in the tail fin neural crest, and since neural crest isrequired for growth of the fin, noggin may act as a growth factor forepidermis or mesenchyme.

Although much of our experimental work has involved rescue of embryonicdevelopment, because expression in the notochord persists in the growingtail bud and a discontinuous line of stained cells (indicatingexpression of noggin initiated at new sites) runs the length of the roofplate of the neural tube (and is also apparent in the head mesoderm), webelieve noggin is expressed as an adult cell function also.

A number of applications for noggin are suggested from its properties.

The noggin cDNA should be useful as a diagnostic tool (such as throughuse of antibodies in assays for proteins in cell lines or use ofoligonucleotides as primers in a PCR test to amplify those with sequencesimilarities to the oligonucleotide primer, and to see how much nogginis present, e.g. primers such as 5' Primers 1-3 and 3' Primers 1-3).

Because noggin has a pattern of expression that suggests it is used toregulate cartilage production in the embryonic head, clinical uses toregulate cartilage and bone growth are suggested for noggin intherapeutic compositions and particularly in combination with othergrowth factors due to a property of noggin to potentiate at least somegrowth factors. Since neural crest cells are required for the tadpolefin to grow, noggin seems to be a growth factor for the tissue matrixand epidermis and should prove useful, for example, in wound healingcompositions.

Noggin, of course, provides the key to isolate its receptor Since manyreceptors mutate to cellular oncogenes, the noggin receptor should proveuseful as a diagnostic probe for certain tumor types. Thus, when oneviews noggin as ligand in complexes, then complexes in accordance withthe invention include antibody bound to noggin, antibody bound topeptides derived from noggin, noggin bound to its receptor, or peptidesderived from noggin bound to its receptor. Mutant forms of noggin, whichare either more potent agonists or antagonists, are believed to beclinically useful. Such complexes of noggin and its binding proteinpartners will find uses in a number of applications.

Practice of this invention includes use of an oligonucleotide constructcomprising a sequence coding for noggin and for a promoter sequenceoperatively linked to noggin in a mammalian, bacterial or a viralexpression vector. Expression and cloning vectors contain a nucleotidesequence that enables the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomes,and includes origins of replication or autonomously replicatingsequences. The well-known plasmid pBR322 is suitable for most gramnegative bacteria, the 2 μ plasmid origin for yeast and various viralorigins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloningvectors in mammalian cells.

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. Typically, this is a gene that encodes aprotein necessary for the survival or growth of a host cell transformedwith the vector. The presence of this gene ensures that any host cellwhich deletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate or tetracycline, (b) complementauxotrophic deficiencies.

Examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR) or thymidine kinase. Such markers enablethe identification of cells which were competent to take up the nogginnucleic acid. The mammalian cell transformants are placed underselection pressure in which only the transformants are uniquely adaptedto survive by virtue of having taken up the marker. Selection pressureis imposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed.Amplification is the process by which genes in greater demand for theproduction of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.Increased quantities of noggin can therefore be synthesized from theamplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumwhich contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell in this case is the Chinese hamster ovary (CHO)cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub and Chasin, Proc. Nat. Acad. Sci., 77, 4216 (1980).The transformed cells then are exposed to increased levels of Mtx. Thisleads to the synthesis of multiple copies of the DHFR gene and,concomitantly, multiple copies of other DNA comprising the expressionvectors, such as the DNA encoding noggin. Alternatively, host cellstransformed by an expression vector comprising DNA sequences encodingnoggin and aminoglycoside 3' phosphotransferase (APH) protein can beselected by cell growth in medium containing an aminoglycosidicantibiotic such as kanamycin or neomycin or G418. Because eukaroticcells do not normally express an endogenous APH activity, genes encodingAPH protein, commonly referred to as neo resistant genes, may be used asdominant selectable markers in a wide range of eukaryotic host cells, bywhich cells transformed by the vector can readily be identified.

Expression vectors, unlike cloning vectors, should contain a promoterwhich is recognized by the host organism and is operably linked to thenoggin nucleic acid. Promoters are untranslated sequences locatedupstream from the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofnucleic acid under their control. They typically fall into two classes,inducible and constitutive. Inducible promoters are promoters thatinitiate increased levels of transcription from DNA under their controlin response to some change in culture conditions, e.g. the presence orabsence of a nutrient or a change in temperature. At this time a largenumber of promoters recognized by a variety of potential host cells arewell known. These promoters can be operably linked to noggin encodingDNA by removing them from their gene of origin by restriction enzymedigestion, followed by insertion 5' to the start codon for noggin.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein which participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist then synthetic oligonucleotide adapters or linkers are usedin accord with conventional practice.

Transcription of noggin-encoding DNA in mammalian host cells iscontrolled by promoters obtained from the genomes of viruses such aspolyoma, cytpmegalovirus, adenovirus, retroviruses, hepatitis-B virus,and most preferably Simian Virus 40 (SV40), or from heterologousmammalian promoters, e.g. the actin promoter. Of course, promoters fromthe host cell or related species also are useful herein.

In particular embodiments of the invention expression of noggin in E.coli is preferably performed using vectors which comprise the following:a lac UV5 promoter which may be controlled by the lactose operonrepressor; a strong ribosome binding site, for example, the ribosomebinding site of bacteriophage T7; a mutation in the replication controlregion of the plasmid which may increase copy number; and a mutationwhich limits the expression of the antibiotic resistance protein.

In a preferred embodiment, noggin is expressed in a high copy numberkanamycin resistant pBR322-derived plasmid under the control of a lacUV5 promoter. In an additional preferred embodiment, noggin is expressedin baculovirus under the control of the polyhedrin promoter ofAutrographa californica Multiple Nuclear Polyhedrosis virus in insecthost cells.

Noggin is believed to find use as an agent for enhancing the survival orinducing the growth of nerve and muscle cells. It, therefore, is usefulin the therapy of congenital conditions or degenerative disorders of thenervous system ("neurodegenerative diseases"), including such diseasesas Alzheimer's disease, Parkinson's disease, Huntington's chorea, ALS,peripheral neuropathies, and other conditions characterized by necrosisor loss of neurons, whether central, peripheral, or motorneurons. Inaddition, it may be useful for treating damaged nerve cells, e.g.,nerves damaged by traumatic conditions such as burns and wounds,diabetes, kidney dysfunction, and the toxic effects of chemotherapeuticsused to treat cancer and AIDS. It also is useful as a component ofculture media for use in culturing nerve cells in vitro.

The capacity of noggin to induce neural tissue may be useful in diseaseswhere neural tissue is formed improperly or incompletely duringdevelopment. Thus, noggin and the noggin gene are also useful intreating congenital malformations such as anencephaly, or the loss ofcerebral hemispheres which results from failure of closure of theanterior neural tube during development. Practice of this inventionincludes preparation and uses of a diagnostic or therapeutic agentcomprising a nucleotide sequence of at least about 15 DNA or RNA basesanalogous to all or a portion of either SEQ ID NO:8, SEQ ID NO:10, SEQID NO:25, or SEQ ID NO:1 or of the nucleic acid sequences contained inbacteriophages, hnogλ-9 or hnogλ-10. That is, noggin preparations areuseful as standards in assays for noggin and in competitive-typereceptor binding assays when labelled with radioiodine, enzymes,fluorophores, spin labels, and the like. Therapeutic formulations ofnoggin are prepared for storage by mixing noggin having the desireddegree of purity with optional physiologically acceptable carriers,excipients or stabilizers, in the form of lyophilized cake or aqueoussolutions. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin or immunoglobulins. Other components can include glycine,blutamine, asparagine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or PEG.

Noggin may be used according to the invention as described supra. Theconcentration of the active ingredient used in the formulation willdepend upon the effective dose required and the mode of administrationused. The dose used should be sufficient to achieve circulating plasmaconcentrations of active ingredient that are efficacious. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems.

By referring to noggin, the present invention also contemplates the useof fragments, derivatives, agonists or antagonists of noggin molecules.

Noggin may be administered in any pharmacologically acceptable carrier.The administration route may be any mode of administration known in theart, including but not limited to intravenously, intrathecally,subcutaneously, by injection into involved tissue, intraarterially,intranasally, orally, or via an implanted device. The present inventionprovides for pharmaceutical compositions comprising noggin in apharmacologically acceptable carrier.

Administration may result in the distribution of noggin throughout thebody or in a localized area. For example, in some conditions whichinvolve distant regions of the nervous system, intravenous orintrathecal administration of noggin may be desirable. Alternatively,and not by way of limitation, when localized regions of the nervoussystem are involved, local administration may be desirable. In suchsituations, an implant containing noggin may be placed in or near thelesioned area. Suitable implants include, but are not limited to,gelfoam, wax, or microparticle-based implants.

Depending upon the mode of administration, the active ingredient may beformulated in a liquid carrier such as saline, incorporated intoliposomes, microcapsules, polymer or wax-based and controlled releasepreparations, or formulated into tablet, pill or capsule forms.

Inventive complexes comprise a ligand characterized by one or more ofthe SEQ ID NOS:3-7. The ligand can be bound to a protein, such asantibody. Such antibodies can be polyclonal or monoclonal. Polyclonalantibodies to noggin generally are raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of noggin and anadjuvant. It may be useful to conjugate noggin or a fragment containingthe target amino acid sequence to a protein which is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example, maleimidobenzoyl sulfosuccinimideester (coniugation through cysteine residues), N-hydroxy-succinimide(through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹ N=C=NR.

Animals can be immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally in multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFruend's complete adjuvant by subcutaneous injection at multiple sites.Seven to 14 days later animals are bled and the serum is assayed foranti-noggin titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the same nogginpolypeptide, but conjugated to a different protein and/or through adifferent cross-linking agent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

Monoclonal antibodies are prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g. by fusion with myeloma cells or by EB virus transformation andscreening for clones expressing the desired antibody.

In a preferred embodiment, a rat monoclonal antibody such as RP57-16,prepared after immunization of a rat with recombinant human noggin,reacts specifically with both Xenopus and human noggin, but not with theneurotrophins BDNF, NT-3 and NT-4.

Noggin antibodies are useful in diagnostic assays for noggin or itsantibodies. In one embodiment of a receptor binding assay, an antibodycomposition which binds to all of a selected plurality of members of thenoggin family is immobilized on an insoluble matrix, the test sample iscontacted with the immobilized antibody composition in order to adsorball noggin family members, and then the immobilized family members arecontacted with a plurality of antibodies specific for each member, eachof the antibodies being individually identifiable as specific for apredetermined family member, as by unique labels such as discretefluorophores or the like. By determining the presence and/or amount ofeach unique label, the relative proportion and amount of each familymember can be determined.

Noggin antibodies also are useful for the affinity purification ofnoggin from recombinant cell culture or natural sources. Nogginantibodies that do not detectably cross-react with other growth factorscan be used to purify noggin free from these other family members.

Aspects of the invention will now be illustrated by the followingexamples.

EXPERIMENTAL PROCEDURES

Production of Xenopus embryos

Xenopus embryos were prepared by the protocol described by Condie andHarland (Development, 101, 93-105, 1987). Embryos were staged accordingto the table of Nieuwkoop and Faber ("Normal Table of Xenopus laevis"(Daubin), Amsterdam: North Holland, 1967). Ventralized embryos wereproduced by irradiation with a Statalinker (Stratagene), and dorsalizedembryos were produced by treatment with LiCl as described by us in ourpaper on certain "wnt" proteins (designated "Xwnt-8" ), Smith andHarland, Cell, Vol. 67, pp. 753-765 (1991) (incorporated by referenceand occasionally referred to hereinafter as "S&H, supra").

EXAMPLE 1

Isolation and Sequencing of Noggin cDNA

The construction of the size-selected plasmid cDNA library from stage 11LiCl-treated embryos was as follows. Sixty micrograms of poly(A) RNAfrom stage 11 LiCl-treated embryos was size fractionated on a 10% to 30%sucrose gradient in the presence of methylmercuric hydroxide. Firststrand cDNA was synthesized from 2 μg of the size-fractionated poly(A)RNAs primed with oligo(dT) oligonucleotide containing the recognitionsite for NotI. After synthesis of the second strand, cDNAs were treatedwith EcoRI methylase, ligated with EcoRI linkers, digested with EcoRIand NotI, and finally ligated to 125 ng of modified pGEM-5Zf(-)(Promega). The pGEM-5Zf(-) used here was modified by the addition of anoligonucleotide into the Nsil site to create an EcoRi site. The vectorwas not treated with alkaline phosphatase, but the excised polylinkersequence was removed on a sepharose 4BCL column. The ligated productswere used to transform XL-I Blue cells (Stratagene), and plated to give100,000 colonies per 10 cm plate. Plasmid DNAs were isolated from platecultures by the alkaline-lysis/polyethylene glycol precipitationprotocol.

Dorsalizing activity in the library was assayed by injecting RNAtranscripts made from pooled plasmid DNA. Single clones were isolated bya process of sib selection. In this procedure the plasmid library wasreplated on 12 plates with 10-fold fewer colonies per plate. RNA wassynthesized from pooled plasmid DNAs isolated from each plate and testedfor dorsalizing activity by injection into UV-ventralized embryos. Thosepools with dorsalizing activity were replated and screened as describedabove. This process was repeated until single clones were isolated.

In vitro RNA synthesis, injection assay for dorsal axis rescue andsib-selections were also done, as described by us in S&H, supra.

The nucleotide sequence of both strands of the isolated noggin cDNAclone was determined by the dideoxy termination method using modified T7DNA polymerase (US Biochem). Deletions were prepared in sequencingtemplates by both restriction enzyme and exonuclease III digestion(Henikoff, Meth. Enzymol, 155, 156-165, 1987).

In vitro translation

One-half μg of in vitro synthesized noggin, Xwnt-8, and goosecoid mRNAswere translated in a nuclease treated rabbit reticulocyte lysate(Promega) with added ³⁵ S-methionine according to the manufacturer'sinstructions. The translation products were visualized bySDS-polyacrylamide gel electrophoresis (12% gels) followed byfluorography. Noggin protein had the molecular weight predicted by theopen reading frame.

RNA Isolation and Analysis

Total RNA was isolated from embryos and oocytes by a small scaleprotocol as described by Condie and Harland, supra. Dorsal lips weredissected from 30 unfixed stage 10.5 embryos and pooled for total RNApreparation. Samples containing either the total RNA equivalent of 2.5embryos or approximately 2 μg of poly A+ RNA were analyzed by northernblotting. Random primed DNA probes were prepared from a 1,323 bpfragment of noggin CDNA from the EcoRI site at nucleotide -83 to anEcoRV site that lies in the vector immediately 3' to the end of thecDNA.

RNAse protection assays were done using a protocol as detailed by Meltonet al. (Nuc. Acids Res., 12, 7035-7056, 1984) with minor modifications(C. Kintner, Salk Institute, La Jolla, Calif.). A noggin cDNAexonuclease III deletion clone, illustrated by SEQ ID NO:8 but having adeletion from the 3' end to nucleotide 383, was used as a template forsynthesizing RNA probes. The template DNA was linearized by EcoRIrestriction enzyme digestion and a 463 base antisense RNA incorporating³² P was synthesized with T7 RNA polymerase. A 387 base antisense EFlαRNA probe was used as a control for amount of RNA per sample Probes weregel purified prior to use.

In situ hybridization

After fixation and storage, the embryos were checked to ensure theblastocoel and archenteron were punctured. Care was taken to puncturethe residual blastocoel of neurulae and tadpoles as well as thearchenteron. Embryos were rewashed at room temperature in 100% ethanolfor two hours to remove residual lipid. After hybridization, stainingwas allowed to develop overnight and the embryos were then fixed inBouin's. Newly stained embryos have a high background of pink stain butmost of this washes out, leaving the specific stain. Following overnightfixation, the embryos were washed well with 70% ethanol, 70% ethanolbuffered with PBS and methanol. Embryos were cleared in Murray's mix andphotographed with Kodak Ektar 25 film, using a Zeiss axioplan microscope(2.5 or 5× objective with 3×12B telescope to assist with focusing).

Lineage Tracing

Lineage tracing with mRNA that encodes nuclear localized B-galactosidasewas as we described in S&H, supra. Ventralized embryos were coinjectedat the 32 cell stage with 0.5 ng B-galactosidase and 25 pg nogginΔ5'mRNAs. Embryos were fixed and stained with X-gal at approximately stage22.

Results

Noggin cDNA Encodes a Novel Polypeptide

The 1833 nucleotide sequence of the selected clone is shown by SEQ IDNO:8 and sometimes also referred to as "clone A3." The sequence containsa single long open reading frame encoding a 222 amino acid polypeptidewith a predicted molecular weight of 26 kDa. At the amino terminus, thehydrophobic stretch of amino acids suggests that the polypeptide entersthe secretory pathway. There is a single potential site for N-linkedglycosylation at amino acid 61. Extensive untranslated regions arelocated both 5' and 3' to the reading frame (593 and 573 bp,respectively). The 3' untranslated region is particularly rich inrepeated dA and dT nucleotides, and contains, in addition to apolyadenylation signal sequence located 24 bp upstream from the start ofthe poly A tail, a second potential polyadenylation sequence 147 bpfurther upstream.

Sense RNA synthesized from clone A3 with SP6 RNA polymerase wastranslated in a rabbit reticulocyte lysate system. The 3S-labeledproducts were fractionated on a 12% SDS-polyacrylamide gel andvisualized by fluorography. The major protein product had the expectedmolecular weight of approximately 26 kDa.

Comparison of the amino acid sequence of the predicted polypeptide tothe National Center for Biotechnology Information BLAST network(non-redundant data base) did not identify any similar sequence. Thus,this clone encodes the new type of protein we have named "noggin" whichis secreted, and which has dorsal inducing activity in Xenopus.

Noggin mRNA can Rescue a Complete Dorsal-Ventral Axis

Injection of noggin RNA into a single blastomere of a four cell stageUV-ventralized embryo can restore the complete spectrum of dorsalstructures. The degree of axis rescue was dependent upon the amount ofRNA injected, with embryos receiving low doses having only posteriordorsal structures, while embryos receiving higher doses had excessdorsal-anterior tissue. RNA transcripts from two noggin plasmids weretested. The first contained the full cDNA. The second (pNogginΔ5') had adeletion removing the first 513 nucleotides of the 5' untranslatedregion up to the EcoRI site. The resulting embryos from injection of RNAtranscripts of these two plasmids, as well as Xwnt-8 RNA for comparison,were scored according to the dorsoanterior index (DAI) scale of Rao andElinson (Dev. Biol., 127, 64-77, 1988). In this scale, a completelyventralized embryo is scored as zero, a normal embryo is scored as 5,and the most severely dorsoanteriorized embryos, those having radialdorsoanterior structures, were scored as 10. RNA synthesized frompNogginΔ5' (nogginΔ5' mRNA) repeatedly gave a higher DAI than theequivalent amount of mRNA synthesized from the complete cDNA. Thedose-dependency of axis rescue by nogginΔ5' mRNA was very similar tothat of Xwnt-8 mRNA.

UV treated embryos were also injected with a higher doses (1,000 pg) ofthe noggin mRNAs. Injection of this dose of noggin mRNA into oneblastomere at the four cell stage resulted in embryos with very severehyperdorsalization (DAI>7). However, most of these embryos died at thelate gastrula/early neurula stage. Apparently excessively stronggastrulation movements resulted in the thinning and rupture of theblastocoel roof. We have also observed this effect with high doses ofinjected Xwnt-8 mRNA.

The rescue of dorsal development by both nogginΔ5' and Xwnt-8 mRNAsfollowed a consistent pattern in which increasing amounts of the mRNAslead to progressively more anterior structures being rescued. Forexample, embryos that received 1 pg of the RNAs had primarily theposterior and trunk dorsal structures rescued, and for the most partlacked head structures. Higher doses (10 or 100 pg) of both of the RNAsresulted in embryos with more anterior development, and many had eithernearly normal or hyperdorsalized phenotypes.

Noggin Injected Blastomeres Act as a Nieuwkoop Center

The effect of varying the site of noggin mRNA injection wasinvestigated. Thirty-two cell stage UV-treated embryos were injectedwith either 0.5 ng of B-galactosidase mRNA alone or 0.5 ngB-galactosidase mixed with 25 pg nogginΔ5' mRNA. Injection of nogginmRNA into blastomeres of the vegetal tier gave the most stronglydorsoanteriorized embryos. In both of the vegetal injected embryos thenuclear X-Gal staining was found almost exclusively in the endoderm (themRNA encodes a B-galactosidase that translocates to the nucleus,allowing distinction from the diffuse background stain). One of theembryos shown was strongly hyperdorsalized (DAI approximately 7) as aresult of the noggin mRNA injection, and had a severely truncated tailand enlarged head structures. Embryos were also rescued by noggin mRNAinjections into the marginal zone.

In these embryos B-galactosidase staining was observed primarily in theaxial and head mesoderm. Injection of noggin mRNA into the animal polehad very little effect on axis formation. Likewise, B-galactosidase mRNAalone was without effect.

Noggin mRNA is Expressed Both Maternally and Zygotically

In northern blot analysis of RNA from Xenopus embryos two noggin mRNAspecies of approximate sizes 1.8 and 1.4 kb were observed. A relativelylow level of noggin mRNA was detected in oocytes. By stage 11 the levelof noggin mRNA was significantly higher, reflecting zygotictranscription (as opposed to the maternally deposited transcripts seenin oocytes). Noggin mRNA remained at the elevated level up to the lateststage examined (stage 45).

We expect that the primary dorsalizing RNA in our library to be elevatedin LiCl-treated embryos relative to normal or UV-treated embryos.Lithium ion treatment resulted in a large increase in the amount ofnoggin mRNA expressed, relative to untreated embryos.

UV treatment had the opposite effect. Noggin mRNA expression wasessentially undetectable in total RNA samples from these embryos.

Thus, the abundance of noggin mRNA in manipulated embryos parallels therescuing activity.

We analyzed the distribution of noggin in oocytes and cleavage stageembryos. Since the amount of maternally deposited noggin RNA is too lowfor in situ hybridization to detect above background, we used an RNAseprotection assay. Oocytes were dissected into animal and vegetal halves.No enrichment of noggin mRNA was seen in either hemisphere relative tototal oocyte RNA. Four-cell stage embryos were dissected into dorsal andventral halves, as well as animal and vegetal halves. Noggin transcriptswere found to be distributed evenly between dorsal and ventralhemispheres as well as animal and vegetal hemispheres. The same resultwas obtained with embryos that were tilted 90° immediately followingfertilization and then marked with a vital dye on their uppermost sideto indicate the future dorsal side. Older (32 cell stage) blastulaembryos were also dissected into dorsal-ventral and animal-vegetalhalves. No enrichment of noggin mRNA in any of the hemispheres was seenrelative to the total embryo. In addition, treatment did not alter theabundance of maternally deposited noggin RNA, indicating no preferentialdegradation in ventral tissues. Samples with known amounts of in vitrosynthesized noggin mRNA were included in the RNAase protection assay.From these and other data we estimate that there is approximately 0.1 pgof noggin mRNA per blastula stage embryo and 1 pg per gastrula stageembryo.

The localization of noggin transcripts was investigated in earlygastrula stage embryos. Dorsal lips were dissected from stage 10.5embryos. A northern blot of equal amounts of total RNA from intactembryos, dissected dorsal lips, and from the remaining embryo afterdissection of the dorsal lip was hybridized with a noggin probe and thenre-hybridized with an EFla probe, as a control for amount of RNA loadedper sample. The autoradiograph of the blot showed that noggin mRNA atthis stage is enriched in the dorsal lip.

In situ Hybridization: Zygotic Expression of Noggin in the SpemannOrganizer

The localization of noggin transcripts in developing embryos wasexamined in greater detail using whole mount in situ hybridization.Whole fixed embryos were hybridized with digoxigenin containing RNAprobes.

Hybridized RNA probe was then visualized with an alkalinephosphatase-conjugated anti-digoxigenin antibody. The specificity ofhybridization seen with antisense noggin probes was tested both byhybridizing embryos with sense noggin probes, and by using twonon-overlapping antisense probes. Due both to the low level ofexpression, and to background staining, noggin mRNA could not bedetected unequivocally before the late blastula stage. The increasedlevel of noggin mRNA that was detected by northern blot followingactivation of zygotic transcription was apparent in in situhybridization at stage 9 as a patch of staining cells on the dorsal sideof the embryo. Viewed from the vegetal pole, this patch of cells wasrestricted to a sector of about 600. A side view of the same embryoshows that the staining cells were located within the marginal zone(i.e., between the animal and vegetal poles and within the presumptivedorsal mesoderm forming region). Transcripts are largely restricted tothe nucleus at this stage.

A side view of an early gastrula stage embryo 30 (approximate stage10.5) shows specific hybridization primarily in the involuting mesodermat the dorsal lip. A vegetal view of the same embryo (blastopore liparrowed) shows that noggin mRNA is most abundant on the dorsal side, butexpression extends at the lower level to the ventral side of the embryo.This method of in situ hybridization does not detect transcripts in themost yolky endodermal region of embryos, therefore we cannot rule outexpression in more vegetal regions than those seen in the Figure.Treatments which are known to affect the size of the dorsal lip (LiCltreatment, UV irradiation) had a profound effect on the pattern ofnoggin in situ hybridization. In LiCl treated embryos the staining isintense throughout the marginal zone. UV treatment reduced thehybridization signal to low levels. This result is consistent withamounts of noggin mRNA seen by northern blot analysis. The UV treatedembryo also is a negative control for specificity of hybridization.

As gastrulation proceeds, noggin mRNA staining follows the involutingdorsal mesoderm, and is highest in the presumptive notochord. By thelate neurula stage (approximately 18) noggin mRNA expressing cells areclearest in the most dorsal mesoderm, primarily in the notochord butalso extending more anteriorly into the pre-chordal mesoderm. Theanterior tip of the notochord is arrowed. During tailbud stagesexpression of noggin in the dorsal mesoderm declines, through expressionin the notochord persists in the growing tailbud. Expression of noggininitiates at several new sites, which become progressively clearer asthe tadpole matures. A discontinuous line of stained cells runs thelength of the roof plate of the neural tube. Staining is also apparentin the head mesoderm, primarily in the mandibular and gill arches. Wesuspect that this expression corresponds to skeletogenic neural crestcells. Furthermore, subsets of these cells express homeobox genes thatmark different anterior-posterior levels of the head neural crest, forexample En-2 in the mandibular arch is seen by antibody staining. Cellswith stellate morphology stained from noggin mRNA in the tail fin. Thesestellate cells are also likely to be derived from the neural crest. Noneof these patterns were seen with the sense probe, or with a number ofother probes.

EXAMPLE 2

Noggin cDNA Transfected into COS Cells Produces Active ConditionedMedium

For COS cells the noggin cDNA was inserted into a COS cell expressionvector. COS cells were transfected, and medium harvested after allowingexpression of the introduced noggin genes. This medium has been testedin an animal cap assay for mesoderm inducing or dorsalizing activity. Wehave tested two transfection protocols, a standard one, where cellsrecover and then are transferred to serum-free medium, and an alternatewhere cells are transferred to a defined medium lacking serum butcontaining transferrin, insulin, and BSA. Cells remain healthy in thesupplemented medium and a cotransfected β-galactosidase gene gives 100fold more activity than in the unsupplemented medium. The results oftreating cells with these media is shown below in Table 1. Animal capswere taken from ventralized animals, treated and at the end ofneurulation they were scored for elongation, usually a sign thatnotochord or neural tissues have been induced. Elongation is indicatedin Table 1 by a "+" and even greater elongation a "++." In addition,they are scored for a molecular marker by Northern blotting.

As shown by the data of Table 1, the noggin cDNA has a large effect onthe COS cell conditioned medium. However, noggin is probably interactingwith something else in the medium, since COS-cell conditioned mediumalone has some activity. It is possible that noggin is causing the cellsto secrete something that they normally would not, but the experimentsdo indicate that noggin is secreted and is responsible for some of theactivity.

                  TABLE 1                                                         ______________________________________                                        Cos Cell Conditioned Medium: Effects on Animal Caps                           expression          Elongation                                                                             N-CAM                                            ______________________________________                                        Transformed to serum free                                                     medium + transferrin,                                                         BSA, and insulin                                                              1. Vector only      +/-      +                                                2. Noggin cDNA      ++       ++                                               Tranferred to serum free                                                      medium without supplements                                                    1. Vector only      -        -                                                2. Noggin cDNA      -        -                                                ______________________________________                                    

Noggin mRNA Injected into Oocytes Produces Active Secreted NogginProtein

A second approach to studying whether protein can be secreted in activeform is to inject oocytes with mRNA and take material secreted by theoocyte. A particular advantage of this method is that the injected mRNAis efficiently translated, and most of the translation of the oocyte canbe taken up by the injected mRNA. A new protein, whose synthesis isdirected by injected noggin mRNA is secreted into the medium. Nogginclearly synergizes with activin to produce elongated explants thatexpress elevated levels of muscle actin.

Biochemical Properties of Noggin

Injected oocytes are injected with mRNA, and labelled with ³⁵ Smethionine. Most of the radioactive protein secreted into the medium isfrom the injected mRNA. The noggin protein, which is almost isotopicallypure, can then be analyzed. From this analysis we have determined thatnoggin is a dimeric glycoprotein. When run under reducing conditions,and treated with N-glycanase to remove sugar residues, noggin migratesonly slightly slower than its predicted molecular weight of 26 kDa. Theremoval of sugar side chains results in a loss of about 4 kDa from astarting apparent molecular weight of 33 kDa. When run undernon-reducing conditions it migrates at double this value.

We do not yet know if the dimer of the protein is the active species, orif there is a proteolytically processed form which is active. In acontrol experiment with activin mRNA, oocytes produce activin activity,but the bulk of the radiolabelled protein migrates as the precursorform. Only a small amount of processed protein (1:5 kDa) was detected.It is possible that noggin injected oocytes secrete predominantlyunprocessed protein and a trace of extremely active processed proteinthat we have not detected. Despite the caveats, the main point fromanalysis of injected oocytes and transfected COS cells is that activenoggin can be obtained as a freely soluble secreted polypeptide. Thissets it apart from the other group of genes with dorsalizing activity,the wnts. Wnt proteins have not been available in soluble form and thishas greatly hampered the analysis of their biological activities, and ofthe receptor that binds to them.

EXAMPLE 3

Cloning of the Mouse Noggin Homolog

It is currently impossible to eliminate zygotic noggin transcriptionfrom developing Xenopus embryos. In contrast, it should be possible togenerate homozygous null mutations in the mouse. We have cloned themouse noggin cDNA (SEQ ID NO:10). This is useful to generate mutantmice. In addition to generating the probes and tools to make mutantmice, a comparison of the noggin sequences should be a useful predictorof conserved domains and functions. The C-terminal 80 amino acids are87% identical between SEQ ID NOS:8 and 10.

Mouse noggin was isolated from an embryonic cDNA library by probing witha radiolabelled frog noggin cDNA under conditions of moderate stringency(as defined earlier). Subsequently a genomic clone was isolated byprobing a genomic library with the mouse noggin cDNA 15 under conditionsof high stringency (as defined, but hybridized at 42° C. and washed at50° C. in 15 mM NaCl, 1.5 mM sodium citrate). The full nucleotidesequence of mouse noggin cDNA (SEQ ID NO:25) as well as the deducedamino acid sequence (SEQ ID NO:26) are shown in FIG. 13. There are onlytwo amino acid differences between mouse noggin and human noggin.

EXAMPLE 4

Cloning of the Human Noggin Homolog

Materials and Methods

Probe preparation

Two oligonucleotides were synthesized based on the mouse noggin sequence(supra). The sequence of the oligonucleotides is noggin 5':5'-CAG ATGTGG CTG TGG TCA-3' (SEQ ID NO:18) corresponding to amino acids QMWLWS(SEQ ID NO:19) and noggin 3':5'-GCAGGAACACTTACACTC-3' (SEQ ID NO:20)corresponding to amino acids ECKCSC (SEQ ID NO:21) of the mouse nogginprotein.

The oligonucleotides were used for PCR amplification of a segment of DNAof 260 nucleotides using as a template a mouse cDNA clone prepared asset forth in Example 3. The amplified fragment had a nucleotide sequencethat corresponds to nucleotides 2 through 262 of the mouse sequence asset forth in SEQ ID NO:10. After amplification, the PCR reaction waselectrophosed in agarose, gels, the DNA band of 260 nts purified byMagic PCR (Promega), and used as template for the probe labelingreaction. The probe was labeled using a standard PCR reaction(Perkin-Elmer) on 20 ng of DNA template and 0.2 m Curie of alpha32P-dCTP (Du Pont 3000 Ci/mmol) instead of dCTP. Unincorporated labelwas separated from the probes on a G50 NICK column (Pharmacia). Theexcluded volume of the reaction contained a total of 1.8×108 cpm.

In addition, one degenerated oligonucleotide, named noggin D,corresponding to conserved mouse and Xenopus noggin sequences, wassynthesized as follows: Noggin D: 5'-GARGGIATGGTITGYAARCC-3' (SEQ IDNO:22). Noggin D (SEQ ID NO:22) was labeled by kinase reaction using T4polynucleotide kinase and gamma 32P ATP. The labeled oligonucleotide waspurified by NAP5 (Pharmacia) column and used for library hybridization.

Library Screening

A human placental genomic library (Clontech Cat#HL1067J, average insertsize 15 kb) in vector EMBL-3 was plated according to manufacturerspecifications in NM 538 E.coli. Approximately 3 million plaques weretransferred to nitrocellulose filters (BA-85 Schleicher and Schuell) inthree replicas (named A, B and C) and screened according to Maniatis, etal. Sambrook, et a., Molecular cloning a laboratory manual, CSH LabPress, New York (1989)!. The replica filters A and C were hybridized ina buffer containing 0.5M sodium phosphate, pH 7.2, 7% sodium dodecylsulphate, 1% crystalline BSA, 1 mM EDTA, 40 m g/ml denaturated salmonsperm DNA and about 1×106 cpm/ml of the PCR probe (supra). Afterhybridization for 12 h at 65° C., the filters were washed twice at roomtemperature in 2× SSC (30 mM sodium citrate, 0.3M NaCl), 0.1% SDS andthen at 65° C. in 2×SSC, 0.1% SDS for 20 min and exposed to Kodak X-OMATAR film. The filter replica B were hybridized with the labeledoligonucleotide noggin D in 6×SSC , 0.1%, SDS at 51° C. for 12 hfollowed by wash at 2×SCC, 0.1% SDS at room temperature, and in 6×SSC,0.1% SDS at 50° C. and exposed to Kodak X-OMAT AR film. Positive plaquesfrom all replicas were isolated and purified by re-screening as above.Purified positive plaques were suspended in 500 μl SM (100 mM NaCl, 10mM MgSO4×7H2O, 50 mM Tris HCl pH 7.5, 0.01% gelatin). 160 μl of phagesuspension was mixed with 0.5 ml saturated NM538 culture, incubated for20 min at 37° C. and then inoculated into 250 ml LB containing 10 mM MgSO4, 0.2% maltose. The cultures were incubated until cell lysis (7-8 hr)at 37° C. The phage lysates were used for phage DNA purification by theQiagen procedure according to the manufacturers recommendations(Qiagen).

Sequencing

Sequencing was performed by using the Applied Biosystems Model 373Aautomatic sequencer and Applied Biosystems Taq DyeDeoxy™ TerminatorCycle Sequencing Kit.

Results

Filters hybridized to the PCR mouse noggin probes (SEQ ID NOS:18 and 20)showed two strong signals corresponding to phage plaques named hnogλ-9and hnogλ-10. These plaques also hybridized to degenerateoligonucleotide probe nogginD (SEQ ID NO:22) revealed that these clonescorrespond to the human noggin gene. In addition, two other plaquesnamed hnogλ-5 and hnogλ-7 produced slightly weaker signals whenhybridized to the PCR probes. These clones correspond to either humannoggin or related gene(s). All of the human DNA inserts can be excisedfrom the vectors using known restriction sites as described in theliterature regarding each particular library.

A 1.6 kb Sacl fragment from clone hnogλ-9 containing the human noggingene was subcloned and the nucleotide sequence determined as set forthin FIG. 1. The amino acid sequence for human noggin, as deduced from thenucleotide sequence, is also set forth in FIG. 1. The gene or cDNA maybe expressed in various eukaryotic or prokaryotic expression systems toproduce biologically active human noggin protein. It is expected thatthe human protein will exhibit neurotrophic activity similar to thatexhibited by Xenopus noggin protein.

EXAMPLE 5

Tissue Localization of message for human noggin

Materials and Methods

Probe preparation

Probes were prepared as set forth in Example 4. The oligos used are asfollows:

SEQ ID NO:23:5'GAC.TCG.AGT.CGA.CAT.CGC.AGA.TGT.GGC.TGT.GGT.CAC

SEQ ID NO:24:5' CCA.AGC.TTC.TAG.AAT.TCG.CAG.GAA.CAC.TTA.CAC.TCG.G

(The underlined sequence represent mouse noggin sequence; the rest ofthe sequence are tails containing restriction sites for cloning.)

A DNA fragment of approximately 300 bp was obtained by PCR amplificationof a mouse cDNA clone prepared as described in Example 3.

RNA Preparation and Northern Blots

Selected tissues were dissected from Sprague-Dawley rats and immediatelyfrozen in liquid nitrogen. RNAs were isolated by homogenization oftissues in 3M LiCl, 6M urea, as described in Bothwell, et al. 1990(Methods of Cloning and Analysis of Eukaryotic Genes, Boston, Mass.,Jones and Bartlett). RNAs (10 μg) were fractionated by electrophoresisthrough quadruplicate 1% agarose-formaldehyde gels (Bothwell, et al.,1990, Methods of Cloning and Analysis of Eukaryotic Genes, Boston,Mass., Jones and Bartlett) followed by capillary transfer to nylonmembranes (MagnaGraph, Micron Separations Inc.) with 10×SSC (pH7). RNAswere UV-crosslinked to the membranes by exposure to ultraviolet light(Stratalinker, Stratagen, Inc.) and hybridized at 68° C. withradiolabled probes in the presence of 0.5M NaPO₄ (pH 7), 1% bovine serumalbumin (fraction V, Sigma, Inc.) 7% SDS, 1 mM EDTA Mahoudi, et al.,Biotechniques 7:331-333 (1989)!, 100 μg/ml sonicated, denatured salmonsperm DNA. Filters were washed at 68° C. with 3×SSC, 0.1% SDS andsubjected to autoradiography for 1 day to 2 weeks with one or twointensifying screens (Cronex, DuPont) and X-ray film (AR-S, Kodak) at70° C. Ethidium bromide staining of the gels demonstrated thatequivalent levels of total RNA were being assayed for the differentsamples as in Maisonpierre, et al., Science 247:1446-1451 (1990)!.

RNA was prepared from the following human cell lines:

    ______________________________________                                        Neuro-  Neuro-     Hema-    Small Cell                                                                              Cervical                                blastoma                                                                              epithelioma                                                                              topoetic Lung Carcinoma                                                                          Carcinoma                               ______________________________________                                        CHP-134 SK-N-MC    K562     Calu 3    HeLa                                    LA-N-1  CHP-100    U937     SKLu                                              LA-N-5  IARC-EWI   M1       NCl-H69                                           IMR-32  SK-N-LO    TF1      SKMES                                             SHSY5Y  SK-ES      BAF                                                        SKNSH   DADY       B9                                                         SHEP                                                                          ______________________________________                                        Sympathoadrenal                    Pheochromo-                                Precursor Hepatoblastoma                                                                            Medulloblastoma                                                                            cytoma                                     ______________________________________                                        MAH       HEPG2       Madsen       PC12                                                             Med                                                                           U266                                                    ______________________________________                                    

RESULTS

We have amplified a DNA fragment from the mouse noggin plasmid,corresponding to the region conserved between Xenopus and mouse noggin.

The amplified fragment of approximately 300 bp was used as probe tohybridize to northerns, with RNAs prepared from adult and embryonictissues, as well as from various cell lines. Noggin transcript of about2 kb in size was detected in adult rat brain, and in a cell line, SKMES,a small cell lung carcinoma.

Expression of noggin transcripts was examined in various tissues fromrat and mouse at different stages of development and in adult. In themouse, noggin transcripts can be detected in embryos or head from E9 toE12, as well as in newborn brain and adult brain. There was nodetectable signal in peripheral tissues examined except in skeletalmuscle. Abundant level of expression was also found in hippocampalastrocytes isolated from postnatal mouse.

In the rat, noggin transcripts were detectable in embryos or head fromE9 to E18, as well as in brain from P1, P19 and adult brain. In thecerebellum, expression of noggin appeared to be higher in E18 and P1; inthe spinal cord, expression of noggin mRNA peaked at P1. Examination ofnoggin expression in all of the CNS regions, especially the olfactorybulb, midbrain, hindbrain and cerebellum. In the adult, noggin mRNAcould be detected in all CNS regions, especially the olfactory bulb andcerebellum. There also appeared to be low levels in the skin.

EXAMPLE 6

Neural Induction by Noggin

Materials and Methods

Preparation of Xenopus nogain CHO cell conditioned medium

Xenopus noggin CHO conditioned medium was made by selecting for stablytransfected CHO cells. Dihydrofolate reductase (DHFR) deficient CHOparental cells (J. Papkoff, Syntex Research) were transfected with aXenopus noggin expression plasmid containing noggin in tandem with thedihydrofolate reductase gene. Growth in nucleoside free medium was usedto select for successfully transfected cells. Nine colonies oftransfectants were picked and grown up individually. The noggin gene inthese cells was amplified by slowly increasing the dose of methotrexate,an inhibitor of DHFR. The presence of noggin transcripts was firsttested by Northern analysis. Subsequently, two clones, B3 and C3, wereshown to secrete noggin protein, since conditioned medium from theselines was capable of dorsalizing ventral marginal zones. Furthermore, bylabeling B3 cellular proteins with 35S-methionine, noggin protein couldbe identified as a band of about 30 KD on reducing SDS-PAGE, and a bandof 60 kD on non-reducing SDS-PAGE indicating it forms the expecteddimer. These properties matched those of the noggin protein previouslyproduced in Xenopus oocytes supra, (Smith et al., Nature 361, 547-49,1993). B3 conditioned medium was collected in a mixture of 1 part alphaMEM and 9 parts CHO-S-SFMII (Gibco-BRL). The cells were allowed tocondition the medium for 3 days. Control medium from parental cells (CHOdhfr-) was collected identically. Twenty fold concentrated medium wasmade using Centriprep 10 concentrators, where the 20 fold change ismeasured by volume.

Purification of human noggin from COS cells

Human noggin protein was purified by a cationic exchange column. COS/M5cells were transiently transfected with a human noggin expressionplasmid, pCAE11. Cells were allowed to condition DMEM (Specialty Media)for two to three days, after which the medium was removed. Particulatesfrom the medium were removed by a centrifugation step and subsequentpassage through a 0.2 um cellulose acetate filter. This cleared mediumwas pumped onto a MonoS (Pharmacia) column which was washed with severalvolumes 40 mM sodium phosphate (pH 7.3), 150 mM NaCl, 1 mM EDTA.Proteins were then eluted in a linear gradient with 40 mM sodiumphosphate (pH 8.5), 1.8M NaCl, 1 mM EDTA. Noggin protein elutes at 0.8MNaCl and is ≧90% pure by SDS-PAGE.

Xenopus otx isolation

To isolate Xenopus Otx clones a tadpole head cDNA library(Hemmati-Brivanlou, et al., Development 106, 611-617, 1989) was screenedwith a mouse otx cDNA (S-L Ang and Rossant, Toronto) at low stringency.The clones that were picked fell into two classes. One class, which wehave designated otxA, included pXOT21.2, the probe used here. By in situhybridization, transcripts are first detected prior to gastrulation inthe superficial layer on the dorsal side. During neurulation a largeanterior domain expressed the gene, and includes both neural andnon-neural tissues. After a decline in expression in the tailbudtadpole, the gene is reexpressed specifically in the brain and eyes.

Ventral marginal zone assay

Embryo preparation

Xenopus laevis embryos are fertilized and de-jellied as described(Condie and Harland, 1987. Development 101, 93-105), routinely theevening before dissections. Embryos are cultured overnight at 15° C. Thevitelline membrane surrounding each developing embryo is manuallyremoved the following morning at the late blastula stage. Untildissection, the embryos are maintained in 1/3× modified ringers inagarose coated dishes.

Ventral marginal zone dissection

Embryos are oriented with their yolky vegetal hemisphere up so thedorsal side can be identified. The dorsal side of the early gastrula ismarked by the presence of a small arc of dense pigment called the"dorsal lip" which marks the start of involution of dorsal mesoderm. Theventral marginal zone (VMZ) is found directly opposite the dorsal lip,and is dissected. Since the vitelline membrane has been removed, theembryo tends to flatten. Using a specially constructed knife made of aneyebrow, mounted onto a glass pipet with wax, two cuts are made throughthe flattened embryo from the top facing vegetal pole through to theanimal pole. The cuts are made such that they isolate approximately30-60 degrees of the ventral side away from more lateral tissues. Athird cut which is perpendicular to the first two cuts completelyisolates the ventral marginal zone tissue away from the rest of theembryo. This third cut is at the level of approximately two thirds ofthe radius of the embryo from the center. Prior to treatment the VMZ iswashed 1× in the culture medium.

Assay

Approximately between 5 to 10 VMZs are used per assay. The washed VMZsare dropped gently (trying to minimize transfer of liquid) intoeppendorf tubes containing the desired treatment protein medium forassay. The VMZs are allowed to develop to the late neurula or earlytailbud stage as assessed by control whole embryo development. At thistime RNA is isolated from the VMZs and control whole embryos asdescribed (Condie and Harland, ibid). The expression of muscle actin inVMZs indicates a dorsalization event (Lettice and Slack, 1933.Development, 117, 263-72). RNA from each sample is run on aformaldehyde-agarose gel and blotted to gene screen. The blot is thenhybridized with a Xenopus muscle actin probe (Dworkin-Rastl et al.,1986. J. Embryol. exp. Morph. 91, 153-68). Quantitation of dorsalizationcan be carried out by normalizing muscle actin signal to that of theubiquitously expressed EF-1α (Krieg et al., 1989. Devl. Biol. 133,93-100). Quantitation is done using phosphor imaging.

RNase protection assay

RNase protection was carried out as described (D. A. Melton et al.,Nucleic Acids Res 12,7035-56, 1984), with the modification thatdigestion was carried out at room temperature (22° C.) using RNase T1only (Calbiochem 556785) at 10 units/ml. 20-30 animal caps wereharvested for each lane, of this 80% was used for neural markers and 10%for muscle actin and collagen type II. For goosecoid and brachyury 20caps were used. Exposures ranged from 12 hours to 5 days. In all cases,films were preflashed. In cases where a marker was not expressed, theresult was confirmed with greater sensitivity using phosphor imaging.

Results

The development of vertebrate embryos requires several inductiveinteractions. Mesoderm, which eventually forms tissues such asnotochord, muscle, heart, mesenchyme and blood, is induced in theequatorial region of the embryo (Nieuwkoop, Wilhelm Roux' Arch.EntwMech. Org, 162, 341-373, 1969). This inductive event is wellstudied, and there are several candidates for the endogenous inducer(s)including members of the fibroblast growth factor(FGF) family andactivin (Jessell and Melton, Cell 68, 257-70 1992; Sive, Genes Dev 7,1-12, 1993) and TGFb family (Asashima, et al., Roux's Arch. Dev. Biol.198, 330-335, 1990; Asashima, et al., Naturwissenschaften 77, 8, 389-91,1990; Green and Smith, Nature 347, 391-394, 1990; Smith, et al., Nature345, 6277, 729-31, 1990; Thomsen, et al., Cell 63, 485-493, 1990; van,et al., Nature 345, 6277, 732-4, 1990). The use of dominant negativereceptors for both FGF (Amaya, et al., Cell 66, 257-270, 1991) andactivin (Hemmati-Brivanlou and Melton, Nature 359, 609-614, 1992) inXenopus embryos strongly suggests that the signaling pathways activatedby these molecules are essential for proper mesoderm formation.Molecules such as wnts (Christian, et al., Development 111, 1045-1055,1991; McMahon and Moon, Cell 58, 1075-84, 1989; Smith and Harland, Cell67, 753-765, 1991; Sokol, et al., Cell 67, 741-752, 1991) and noggin(Smith, et al., Nature 361, 547-49, 1993) modify the kinds of mesodermmade without inducing mesoderm directly.

In a subsequent induction, the dorsal mesoderm of the Spemann organizersignals nearby lateral mesoderm to take on a more dorsal fate (Dale andSlack, Development 100, 2, 279-95, 1987; Lettice and Slack, Development,117, 263-271, 1993; Spemann and Mangold, Arch. Mikrosk. Anat. EntwMech.100, 599-638, 1924; Stewart and Gerhart, Development 109, 363-372,1990). The only known factor which is expressed in the organizer and canmimic its dorsalizing activity is noggin.

Dorsal mesoderm of the Spemann organizer also signals nearby ectoderm tobecome neural tissue. Neural induction by dorsal mesoderm has beendemonstrated in amphibians (Dixon and Kintner, Development 106, 749-757,1989; Doniach, et al., Science 257, 5069, 542-5, 1992; Hamburger, TheHeritage of Experimental Embryology: Hans Spemann and the Organizer,1988; Kintner and Melton, Development 99, 311-25, 1987; Spemann, Arch.mikrosk. Anat. EntwMech. 100, 599-638, 1938), birds (Kintner and Dodd,Development 113, 1495-1506, 1991; Tsung, et al., Acta Biol exp Sinica10, 69-80, 1965), and recently in mice (Ang and Rossant, Development118, 139-149, 1993). Despite decades of effort, little is known aboutthe molecular nature of the factors responsible for this induction.Among known inducers, activin can promote formation of neural tissue,but this is due to a secondary induction by the dorsal mesoderm thatactivin induces (Green, et al., Development 108, 1, 173-83, 1990; Greenand Smith, Nature 347, 391-394, 1990; Kintner and Dodd, Development 113,1495-1506, 1991). Thus, activin cannot promote formation of neuraltissue when added to gastrula ectoderm; however, such ectoderm remainscompetent to be neuralized by dorsal mesoderm until the end ofgastrulation (Sharpe and Gurdon, Development 109, 765-74, 1990).

Direct Neural Induction by Noggin

Candidates for the endogenous inducer are expected to induce neuraltissue in the absence of dorsal mesoderm. Competent animal cap ectodermfrom late blastula stage embryos (St9) was used to test noggin's neuralinducing capacity. Xenopus noggin protein conditioned medium wascollected from stably transfected CHO cells and twenty fold concentratedmedium was used to treat St 9 animal caps. Markers used in an RNaseprotection assay were N-CAM (Jacobson and Rutishauser, DevelopmentalBiology 116, 524-31, 1986; Kintner and Melton, Development 99, 311-25,1987), a neural cell adhesion molecule, a neural specific isoform ofb-tubulin (Good, et al. Nucleic Acids Res 17, 8000, 1989; Good, et al.,Dev Biol 137, 414-8, 1990; Richter, et al., Proc Natl Acad Sci USA 85,8086-90, 1988) that is expressed in the hind brain and spinal cord, andXIF3, a neurally expressed intermediate filament gene (Sharpe, et al.,Development 107, 701-14, 1989) to assay for neural induction. All thesemarkers are restricted to neural tissue, however, only NCAM is expressedthroughout the nervous system. We found that Xenopus-noggin conditionedmedium induces high levels of N-CAM and XIF3 expression FIG. 2.; lane 8!in treated animal caps, without inducing muscle actin (lane 13)(Dworkin-Rastl, et al., J. Embryol. exp. Morph. 91, 153-168, 1986;Mohun, et al., Nature 311, 716-721, 1984). Control CHO cell mediuminduces neither muscle nor neural tissues (lanes 7, 12). St 9 activintreated animal caps express muscle actin (lane 11) and all three neuralmarkers (lane 6), demonstrating activin's ability to generate neuraltissue indirectly. It is interesting to note that noggin induces verylittle, if any b-tubulin expression, while inducing high levels ofN-CAM, but activin induction has nearly the converse effect.

To determine whether noggin protein is sufficient to induce neuraltissue, COS cells were transfected with pCAE11, a human nogginexpression plasmid, and the conditioned medium was purified by cationexchange chromatography resulting in noggin preparations that were 90%pure FIG. 3.!. Such purified human noggin protein is also able to induceneural tissue in animal caps FIG. 4a., see below!.

We have shown that noggin does not induce muscle in late blastula stageanimal caps, however, it is possible that noggin induces other types ofdorsal mesoderm. To address this concern, we asked whether noggin couldinduce the expression of the early mesoderm markers goosecoid (Blumberg,et al., Science 253, 194-6, 1991; Cho, et al., Cell 67, 1111-20, 1991),a marker of organizer tissue and subsequently head mesoderm orX-brachyury (Smith, et al., Cell 67, 79-87, 1991), which appears to beexpressed in all mesodermal precursors early, and subsequently isexpressed in posterior mesoderm and notochord. Animal caps were treatedat stage 9 and collected at stage 11, when expression of goosecoid andbrachyury in the normal embryo is high. Neither marker is turned on bypurified human noggin (FIG. 4b. lane 5) at a dose with demonstratedneural inducing activity (FIG. 6 lane 15); in contrast animal capstreated in the same fashion with activin show both goosecoid and X-braexpression (FIG. 4b. lane 4) as expected for this mesoderm inducingfactor (Cho, et al., Cell 67, 1111-20, 1991; Smith, et al., Cell 67,79-87, 1991). Untreated animal caps show no expression of thesemesodermal markers (lane 3), and RNA levels in the collected animal capsare shown to be comparable using EF-1a levels (Krieg, et al., Dev Biol133, 93-100, 1989).

Since purified human noggin is capable of driving neural induction, noadditional factors which may have been present in the crude conditionedmedium are required. Furthermore, Xenopus and human noggin, with 80%amino acid identity, can both act to induce neural tissue in Xenopus,suggesting a conserved function for these two proteins. However, fornoggin to be a candidate endogenous neural inducer it must be able toinduce neural tissue at a stage when neural induction occurs in normalwhole embryos. It is unclear when the first instructive signals are sentfrom dorsal mesoderm to ectoderm in embryos. However, it is known thatby early gastrula stages, dorsal ectoderm has already been specified tobecome neural tissue (Jones and Woodland, Development 107, 785-91,1989). The neural inducing signal is therefore likely to start beforethis stage. The latest stage at which animal caps have been shown to becompetent to respond to neural-inducing mesoderm is the early neurula(St13-14) (Sharpe and Gurdon, Development 109, 765-74, 1990). Thus, acandidate endogenous neural inducer must be able to induce neural tissuefrom gastrula stage competent ectoderm.

Neural Induction at the Gastrula Stage

In order to assess the competence of ectoderm to respond to noggin wetreated animal caps taken from blastula (St8), late blastula (St9),early gastrula (St10) and ventral animal caps from mid-gastrula (St10.5)stage embryos with purified human noggin FIG. 2.!. We also treatedsimilarly staged animal caps with activin to demonstrate its mesoderminducing and secondary neural inducing activities, and to contrastactivin's effects with those of noggin FIG. 4a.!. Activin treated animalcaps show neural induction only in conjunction with induction of dorsalmesoderm, such as muscle and notochord (lanes 3, 6, 9). In a number ofexperiments, we confirmed that activin's ability to induce dorsalmesoderm, and consequently neural tissue, declines rapidly at thegastrula stage (lane 12) (Green, et al., Development 108, 173-83, 1990;Kintner and Dodd, Development 113, 1495-1506, 1991). In the experimentshown here a larger than usual dose of activin was given. Under theseconditions, only a small amount of neural tissue is made, perhapsbecause so much mesoderm is induced that there is not much competentectoderm left in the explant to be neuralized. In contrast noggin caninduce neural tissue in animal caps taken from all of these stageswithout inducing the notochord and somite marker, collagen type II(Amaya, et al., Development 118, 477-87, 1993; Bieker andYazdani-Buicky, J Histochem Cytochem 40, 1117-20, 1992), or muscle actin(lanes 4, 7, 10, 13). This gives additional support to the proposal thatnoggin is a direct neural inducer, since it can act in the absence ofboth early and late mesoderm markers. Furthermore, we have shown thatnoggin can induce neural tissue in competent ectoderm at a time whenmesoderm inducers are inactive.

In some experiments, noggin addition to gastrula (but not blastula)animal caps resulted in induction of muscle (data not shown). Thisoccurred at stages when activin could no longer induce muscle. Weinterpret this as a result of a dorsalizing action by noggin on tissuesthat have received a weak mesoderm-inducing signal. Themesoderm-inducing signal which spreads into the gastrula animal cap isnot enough to induce mesoderm, but in the presence of Xwnt-8 or noggin,muscle is formed. One interesting corollary of the induction of muscleis that the kinds of neural tissue seen in the explant are modified.Induction in explants that contain no muscle usually yields N-CAMexpression, but if muscle is present, expression of both N-CAM andb-tubulin is seen. This phenomenon is demonstrated in the secondaryneural induction by activin in St. 9 animal caps FIG. 2.! and in thecomparison of neural tissue induced by noggin in ventral marginal zonesversus animal caps FIG. 6.!. In the ventral marginal zones and animalcaps in which muscle is present, both N-CAM and b-tubulin are expressed,whereas induced animal caps without muscle, show only N-CAM expression.

Neural induction after injection of DNA coding for noggin

To confirm our conclusions using a different experimental approach, wehave directed noggin expression to gastrula stage animal caps byinjecting the plasmid pCSKA-noggin into the animal pole of a one cellstage embryo. This plasmid, in which noggin is under the control of thecytoskeletal actin promoter, turns on the expression of noggin mRNA atthe onset of gastrulation (Smith, et al., Nature 361, 547-49, 1993). Atthe blastula stage, the animal caps are dissected and then matured totailbud stages for molecular analysis. Animal caps injected with thenoggin plasmid show expression of N-CAM in the absence of muscle ornotochord markers (FIG. 4c. lane 2). A control plasmid directing theexpression of lac Z showed no neural or mesodermal induction as expected(lane 1). This experiment demonstrates that ectopic noggin expressioncan directly induce neural tissue in gastrula stage ectoderm, a stagewhen neural induction is taking place in whole embryos.

Differences in competence between dorsal and ventral animal caps.

Animal caps taken from the dorsal side of gastrula stage embryos showgreater competence to form neural tissue than ventral animal caps (Otteand Moon, Cell 68, 1021-29, 1992; Sharpe, et al., Cell 50, 749-58,1987), when involuted anterior mesoderm is used as the inducer. Thistype of mesoderm, however, has weaker inducing capacity than the rest ofthe involuted mesoderm (Sive, et al., Cell 58, 171-180, 1989).Furthermore, the ventral side of an embryo can support the formation ofa complete secondary axis when the organizer is placed on that side(Gimlich and Cooke, Nature 306, 471-3, 1983; Smith and Slack, J.Embryol. Exp. Morph. 78, 299-317, 1983; Spemann, Arch. Mikjrosk. Anat.EntwMech. 100, 599-638, 1938), indicating that there is no qualitativedifference in competence. Thus, while a weak inducer might unmask slightdifferences in competence of the ectoderm, it has been suggested that arobust neural inducer would show little difference in its effects ondorsal and ventral ectoderm (Servetnick and Grainger, Development 112,177-88, 1991). Therefore we tested noggin's effects on dorsal andventral ectoderm from the early gastrula. No difference in N-CAMexpression is detected (FIG. 5, lanes 4, 6), while the ventral animalcaps treated with noggin show a greater amount of muscle actinexpression (presumably through dorsalization of tissues that received alow-grade mesoderm induction). Activin treated dorsal caps showinduction of roughly the same level of muscle actin expression (lane 5)as the ventral noggin treated caps, however, activin treatment did notinduce detectable neural specific transcripts (lanes 3, 5). Thisindicates that muscle tissue induced at this stage is not sufficient tosecondarily induce neural tissue, and that noggin must be present toinduce neural tissue.

We conclude that there is no dorsal-ventral difference in nogginmediated neural induction, suggesting that noggin behaves like therobust neural inducing signal of the Spemann organizer, not like theweaker signal from early anterior mesoderm.

Dose Dependence

To determine what levels of noggin protein are required for neuralinducing activity, we carried out a dose response experiment. Inaddition to determining the doses required for neural induction inanimal caps, we have also carried out a dose response of thedorsalization of ventral marginal zones in order to compare the dosesrequired for these two types of inductions. Stage 9 animal caps or St.10.5 VMZ were treated with purified human noggin, and N-CAM andβ-tubulin were used to assay neural induction, while muscle actin wasused as a marker of dorsal mesoderm. This experiment shows that neuralinduction occurs at a dose of 1 μg/ml, which is a twenty fold higherdose than required for dorsalization of VMZ FIG. 5!. There are severalobservations that may account for the apparently high dose requirement.First, to get a maximal neural response from dorsal mesoderm, thetissues must be left in contact through most of neurulation (Sharpe andGurdon, Development 109, 765-74, 1990); in contrast, the animal capstreated with noggin close up rapidly, this inhibits factor access, andconsequently they receive only a brief effective dose. Second, it islikely that noggin is not the only neural inducer active in the embryo;it has been shown in a variety of amphibians that the somites(Hemmati-Brivanlou, et al., Science 250, 800-802, 1990; Jones andWoodland, Development 107, 785-91, 1989) and the neural plate haveneural inducing activity (Hamburger, The Heritage of ExperimentalEmbryology: Hans Spemann and the Organizer, 1988; Servetnick andGrainger, Dev Biol 147, 73-82, 1991) and noggin transcripts are notdetected there. Thus it is plausible that noggin is one of severalneural-inducing activities. In this connection it is worth noting thatnoggin is equally potent in inducing neural tissue in ventral marginalzones as in dorsalizing them to generate muscle. Numerous otherexperiments (see FIG. 5) show that induction of a similar amount ofmuscle at this stage by activin does not result in neural induction.Fourth, it may be that only a small fraction of the purified protein isactive, and that the experiment overestimates the amount of proteinneeded for neural induction. Finally, it is possible that theaccessibility of exogenously added soluble noggin is significantly lowerthan noggin protein being secreted endogenously.

Patterning

Embryonic neural tissue develops an anteroposterior (A-P) pattern, withvarious brain structures, eyes, and the spinal cord. It is thought thatA-P neural pattern requires the presence of dorsal mesoderm, whether itbe adjacent to the responding ectoderm in a planar configuration (Dixonand Kintner, Development 106, 749-757, 1989; Doniach, et al., Science257, 542-5, 1992; Kintner and Melton, Development 99, 311-25, 1987; Ruizi Altaba, Development 108, 595-604, 1990), or directly beneath it in avertical interaction (Dixon and Kintner, Development 106, 749-757, 1989)(Hemmati-Brivanlou, et al., Science 250, 800-802, 1990; Sharpe andGurdon, Development 109, 765-74, 1990; Sive, et al., Cell 58, 171-80,1989). Both of these types of interactions occur in normal development,and both probably contribute to the resulting pattern. To determine ifnoggin induces patterned neural tissue, and if so, what neural regionsare represented, we used Xenopus otx as a marker of forebrain and midbrain; En-2 (Hemmati-Brivanlou, et al., Development 111, 715-724, 1991)as a marker of the mid brain-hind brain boundary, and Krox-20(Wilkinson, et al., Nature 337, 461-4, 1989) as a marker of the thirdand fifth rhombomeres of the hind brain in in situ hybridization(Harland, Methods in Cell Biology, 36, 675-685, 1991). Antibodiesdirected against XlHbox 6 (Wright, et al., Development 109, 225-34,1990) mark posterior hind brain and spinal cord structures. Prior to theuse of these markers, we observed the formation of cement glands innoggin treated animal caps. Since cement glands are induced organs ofectodermal origin found anterior to the neural plate, this resultsuggests that noggin induces anterior structures. In situ hybridizationconfirms this by showing the presence of a cement gland specifictranscript, XAG-1 (Sive, et al., Cell 58, 171-180, 1989) in noggintreated animal caps, but not in control treated animal caps FIG. 7.!. Insitu hybridization with the region specific neural markers FIG. 7.! showthat noggin induces forebrain type tissue as seen by the expression ofotx in noggin treated animal caps. We have not detected En-2, Krox20, orXlHbox, suggesting that these more posterior markers are not induced bynoggin.

Expression of neural antigens

We have demonstrated that noggin directly induces the expression ofneural specific transcripts. A further demonstration is to useantibodies against neural specific antigens to show that the noggininduced tissue is phenotypically neural. To this end, we have treatedanimal cap tissue with noggin and cultured them to a late stage (St 35)for antibody staining. We have used the 6F11 anti-N-CAM antibody, whichstains the entire neural tube of a normal embryo. Noggin treated animalcaps express this antigen FIG. 7.! while control untreated animal capsdo not. This indicates that noggin can induce the production of neuralspecific proteins in treated animal caps. We have failed to detect theexpression of numerous other antigens that are characteristic of varioussubclasses of differentiated neural cells. These included 2G9, whichstains most neural tissue, including peripheral neurons, Tor 24.55,which stains sensory neurons, and Tor 23, which stains a variety ofneurons including motor neurons.

EXAMPLE 7

Production of recombinant human noggin from E. coli and baculovirus

Materials and Methods

Genetic Engineering and Cell Culture

A lactose inducible expression plasmid was constructed by replacing theSwa1/Bsm1 region of pRPN40 (Masiakowski et al, J. Neurochem. 57,1003-1012, 1991) with the Swa1/Bsm1 region of the human noggin geneobtained by PCR and spanned by the same restriction sites, resulting inplasmid pRG301. pRG301 is a high copy number kanamycin resistant plasmidderived from pBR322 with the human noggin gene under the control of thelacUV5 promoter. A plasmid containing the high copy number kanamycinresistant gene was deposited with the Agricultural Research Collection(NRRL), Peoria, Ill., and bears accession number B-18600. This plasmidwas described in U.S. patent application Ser. No. 07/478,338, now U.S.Pat. No. 5,256,568, which is incorporated by reference herein in itsentirety. E. coli W31 10laclq cells transformed with pRG301 were grownat 37° C., induced with lactose, harvested by centrifugation, washedonce with 100 mM Tris-HCl, 50 mM EDTA pH 8 and stored frozen,essentially as described (Masiakowski et al, ibid.).

Recovery from inclusion bodies

E. coli cell paste (32 g) was suspended in ten volumes (v/w) of 50 mMTrisHCl-pH 8.0-5 mM EDTA, lysed in a French Press at 8,000 psi and 8° C.and centrifuged at 8,000×g for 30 min at 40° C. The pellet containingnoggin was suspended in the original volume of 2M urea-20 mM TrisHCl, pH8.0 and stirred for 30 min. The suspension was centrifuged at 8,000×g at4° C. for 30 min and the pellet consisting mostly of inclusion bodies(IB) was suspended in 20 volumes (v/w) of 6M guanidine HCl, 50 mMTrisHCl, 1 mM EDTA, 50 mM DTT and stirred for one hour at roomtemperature. After centrifugation at 8,000×g for 30 min, the supernatantcontaining 0.45-0.50 g denatured and reduced noggin was diafilteredagainst 10 volumes of 6M urea-50 mM sodium acetate pH 4.5-1 mM EDTA-0.1mM DTT using Omega 10,000 MW cut-off membranes. The diafiltratecontaining 0.4-0.44 g noggin was loaded at a flow rate of 30 ml/min ontoa 2.6×10 cm column of S-Sepharose (Pharmacia), equilibrated in 6Murea-50 20 mM sodium acetate-1 mM EDTA-0.1 mM DTT pH 4.5. The column wasfirst washed with the same buffer and then with a one liter gradient(0-1M NaCl) at a flow rate of 30 ml/min. Fractions containing nogginwere identified by gel electrophoresis and pooled. The yield was0.2-0.25 g noggin.

Refolding

The denatured and reduced noggin solution was adjusted to 0.05-0.2 mg/mlprotein concentration and brought to 1.5-2.5M guanidine HCl-0.1M TrisHClpH 8.0-0.1 mM EDTA-0.2-2 mM reduced glutathione-0.02-0.2 mM oxidizedglutathione (preferably at a ratio of 10:1 reduced to oxidizedglutathione) at 4° C. under slow stirring. After 24-72 hours, tworefolded noggin isoforms were identified by RP-HPLC chromatography (FIG.8). The refolded noggin solution was diafiltered against 20 volumes of0.05M sodium acetate pH 4.5, brought to 50 mM potassium phosphate pH 7.2and stirred slowly at 4° C. for 1 hour minimum. Misfolded nogginprecipitated and was removed by centrifugation for 30 min at 8,000×g.

Reverse phase HPLC chromatography.

Refolded noggin can be purified by chromatography on a 12 mm C8, 1×25 cmDynamax 300 A column equilibrated in solvent A (0.1% TFA in water).After loading, the column was washed with solvent A and was developed ata flow rate of 4 ml/min according to the following protocol: (a) 10 minisocratically at 70% of solvent A, 30% of solvent B (0.1% TFA inacetonitrile); 30 min linear gradient to 60% solvent B and 40% solventA. Correctly refolded noggin elutes earlier at 44%-46% solvent B. Theyield was 0.07-0.1 g noggin.

Production of human noggin in Baculovirus cell culture.

The SF21 line of Spodoptera frugiperda was routinely maintained as cellmonolayers in Grace's Insect Cell medium supplemented with lactalbuminhydrolysate and yeastolate (Gibco). This medium completed with 10% v/vheat-inactivated fetal calf serum (Irvine Scientific) is identified asTMNFH-10. Cells were also cultured in serum-free medium (SF-900-II;Gibco) after adaptation. Suspension cultures in either medium wereraised in microcarrier culture flasks (Bellco) using a stirring speed of80 rpm. All cultures were maintained at >96% viability, as judged bytrypan blue exclusion.

Construction of recombinant baculovirus.

Sequences corresponding to human noggin were excised as a5'-BamH1--Pst1-3' fragment from an expression plasmid containing thehuman noggin gene. This fragment was inserted into BamH1--Pst1 digestedpVL1393 (Invitrogen). The resulting plasmid, pTR 1009, has the humannoggin sequence immediately downstream of the polyhedrin promoter ofAutrographa californica Multiple Nuclear Polyhedrosis Virus (AcMNPV),and this promoter-heterologous gene fusion is flanked in turn byrecombination targets derived from the AcMNPV polyhedrin region.Recombinant plasmid DNA was purified by alkaline lysis and CsClcentrifugation. SF21 cells were cotransfected with plasmid and viral DNAby the following method: Plasmid DNA (3 mg) was mixed with 0.5 mglinearized, deleted viral DNA (Baculo Gold™, Pharminigen), andprecipitated with ethanol. Dried DNA was then resuspended in water (50ml), mixed with 1.5 ml Grace's medium, and 30 ml Lipofectin™ cationicliposomes (BRL). The DNA-liposome mixture was vortexed, allowed to standat room temperature for 15 minutes and added dropwise to a monolayer ofSF21 cells (2×106 cells/60 mm plate). After incubation at 27° C. forfour hours, 2 ml TMNFH-10 was added and the culture returned toincubation for 5 days. Tissue culture medium was harvested and used as asource of virus for plaque isolation.

Recombinant virus was isolated by multiple rounds of plaque purificationon SF21 cells. Diluted virus (0.5 ml) was adsorbed to cell monolayers(2×106 cells/60 mm plate) for a period of one hour at 27° C., aspirated,and virus plaques were allowed to develop with an overlay of 0.5%agarose in TMNFH-10 medium for a period of 6 days. Virus plaques werepicked after microscopic inspection, and eluted into 2 ml SF900-IImedium. Virus stocks were amplified by low multiplicity (0.1 pfu/cell)infection. Virus clones expressing noggin were identified by metaboliclabeling of infected cultures with 35S-methionine and 35S-cysteine andanalyzing total labeled protein by polyacrylamide gel electrophoresisand autoradiography. A labeled protein of the expected apparent Mr of20,000-30,000 was detected by this method in candidate clones but not incontrol cultures.

Expression and purification of baculovirus-derived noggin.

SF21 cells were cultured in suspension flasks to a density ofapproximately 1.8×106/ml in SF900-II medium. Cultures (500 ml) werecollected by centrifugation at 1000×g for 10 min and resuspended in 20ml of growth medium containing 5-10 pfu/cell recombinant virus. Viruswas allowed to adsorb for 1 hour at room temperature with gentle mixing.Infected cells were then diluted to their original volume with freshgrowth medium, and incubated at 27° C. for 3 days. Cells and debris weresubsequently clarified from the growth medium by centrifugation at1000×g for 20 min.

Cell supernatants were brought to pH 8.0, passed through a 0.45 mmMillipak 60 filter and applied to a Fast S column that had beenequilibrated in 25 mM HEPES pH 8.0. The column was washed with the samebuffer and developed with a linear NaCl gradient to remove other mediumcomponents. Noggin eluted from this column at 1M NaCl.

Results

Characterization of human noggin produced in E. coli and in baculovirus

Reverse-phase HPLC chromatography shows that recombinant noggin refoldedand purified from E. coli elutes in a single sharp peak, indicating thepresence of one predominant isoform (FIG. 9).

Electrophoresis on 15% polyacrylamide-SDS-reducing gels shows thatnoggin from either E. coli or insect cells is better than 95% pure andmigrates in a single band corresponding to a protein of 20-30 kD. Nogginfrom insect cells shows slightly slower mobility, apparently due toadditional mass from N-linked glycosylation at the single consensus site(FIG. 10). Treatment with Endo F converts the mobility ofinsect-produced noggin to that of the bacterially produced protein (datanot shown).

In the absence of reducing agents, noggin produced either in E. coli orin baculovirus behaves as a disulfide-linked oligomeric protein (FIG.10). However, by gel filtration analysis and mass spectroscopy noggin isprimarily a dimeric protein (data not shown).

Circular dichroism studies show that recombinant noggin refolded andpurified from E. coli as well as noggin purified from insect cells havevery similar conformations (FIG. 11). Secondary structure determined bythis method indicates that noggin consists of 48% alpha-helix, 0%beta-structure, and 52% random coil.

Biological Activity of human noggin produced in E. coli and inbaculovirus

Biological activity of human noggin produced in E. coli or inbaculovirus was determined by assay of muscle actin expression in theventral marginal zone assay, as described supra. Results shown in FIG.12 indicate a positive dose response for induction of muscle actin mRNAin VMZ exposed to either bacterially produced human noggin, orbaculovirus produced human noggin.

EXAMPLE 8

Production and characterization of rat monoclonal antibody RP57-16reactive with human noggin.

Materials and Methods

Production of antibody

RP57-16 rat monoclonal antibody reactive with recombinant human andXenopus noggin was produced by the immunization of a female Lewis ratwith four 35 μg injections of purified recombinant human noggin(produced in E. coli) over a two month period. For the initialimmunization, the protein was injected in the rear foot pad in Freund'scomplete adjuvant. Subsequent injections were given in the same foot padin Freund's incomplete adjuvant. The rat was euthanized 3 days after thefourth injection.

Lymph node cells from the immunized rat were mixed with SP2/0-E.O. mousemyeloma cells at a ratio of 2:1. After centrifugation, the cell mixturewas resuspended in 0.25 ml of 42% (w/v) PEG 3350 (Baker) inphosphate-buffer-saline with 10% (v/v) dimethylsulfoxide (Sigma) for atotal of 3 minutes in a 37° C. water bath. Cells were plated at adensity of 5×10⁴ lymphocytes per well in 96-well plates (Falcon 3072) inDMEM/F-12 (Mediatech, Inc.) containing 10% FBS (supplemented withstreptomycin, penicillin, pyruvate, and glutamine) and HMGT (1.6×10-3Mthymidine, 4.0×10-4 methotrexate, 1.3×10-3 sodium bicarbonate and1.0×10-2 hypoxanthine). After 10 days in culture, supernatants wereharvested and assayed for antibody activity against recombinant humannoggin by indirect ELISA. Supernatant from COS-M5 cells transfected withthe plasmid containing the human noggin gene was air dried overnight inProbind 96-well assay plates (Falcon 3915). Non-specific binding waseliminated by 2 hour incubation at ambient temperature with PBS/1% BSA(Sigma). Plates were washed 2 times, with PBS/0.02% Tween 20. Culturesupernants were then added and incubated at ambient temperature for 1hour. Plates were washed 4 times with PBS/0.02% Tween 20. Secondaryantibody, Goat anti-Rat IgG (H+L) alkaline phosphatase conjugate(Caltag)diluted 1:2000 in PBS/1% BSA was added to each well and the platesincubated at ambient temperature for 1 hour. Plates were again washed 4times with PBS/0.02% Tween 20. Antibody binding was visualized by 1 hourincubation at ambient temperature in the dark with pNPP (pnitrophenylphosphate, Sigma) 1 mg/ml in diethanolamine buffer, pH 9.8. The reactionwas stopped by the addition of an equal volume of 100 mM EDTA.Absorbance was read at 405 nm on a Thermomax Microplate Reader(Molecular Devices). A reaction was considered positive if theabsorbance was 2 times that of the negative control (diluent alonefollowed by secondary antibody and substrate). Positive clones wereexpanded and culture supernatant containing monoclonal antibody wascollected for specificity analysis.

RP57-16 was cloned in soft agar. Cloned hybrid cells were expanded inDMEM/F-12 (Mediatech, Inc.) containing 10% FBS (supplemented withstreptomycin, penicillin, pyruvate, and glutamine). Supernatantcontaining antibody was aliquoted and stored at -70° C. until use.

Specificity Analysis

ELISA

100 ng of purified recombinant human noggin, Xenopus noggin, BDNF, NT-3,and NT4 protein was individually passively adsorbed to Probind 96-wellassay plates by overnight incubation at 4° C. in 50 mM bicarbonatebuffer, pH 9.6. BDNF, NT-3 and NT-4 were used to assess non-specificbinding of rat monoclonal antibody RP57-16. Supernatants from COS-M5cells transfected with either the plasmid containing the human noggingene or the plasmid containing the flg C-terminal tagged Xenopus noggingene were air dried to Probind 96-well plates overnight. Non-specificbinding was eliminated by 2 hour incubation at ambient temperature withPBS/1% BSA (Sigma). Plates were washed 2 times with PBS/0.02% Tween 20.Undiluted RP57-16 was added and incubated at ambient temperature for 1hour. Plates were washed 4 times with PBS/0.02% Tween 20. Secondaryantibody, Goat anti-Rat IgG (H+L) alkaline phosphatase conjugate(Caltag)diluted 1:2000 in PBS/1% BSA was added to each well and the platesincubated at ambient temperature for 1 hour. Plates were again washed 4times with PBS/0.02% Tween 20. Antibody binding was visualized by 1 hourincubation at ambient temperature in the dark with pNPP (p-nitrophenylphosphate, Sigma) 1 mg/ml in diethanolamine buffer, pH 9.8. The reactionwas stopped by the addition of an equal volume of 100 mM EDTA.Absorbance was read at 405 nm on a Thermomax Microplate Reader(Molecular Devices). A reaction was considered positive if theabsorbance was 2 times that of the negative control (diluent alonefollowed by secondary antibody and substrate).

Electrophoresis and Western Blotting

Rat monoclonal antibody RP57-16 was also analyzed by Western blotting.50 ng of recombinant human noggin, non-reduced and reduced, wereelectrophoresed on 12.5% SDS-polyacrylamide gels and electroblotted onnitrocellulose membranes. Membranes were blocked with PBS/1% Casein/0.1%Tween 20, and then incubated for 2 hours with undiluted RP57-16 culturesupernatant. Following 4 washes in PBS/0.02% Tween 20, the membraneswere incubated with a 1:5000 dilution of Goat anti-Rat IgG (H+L)horseradish peroxidase conjugate (Pelfreeze) in PBS/1% BSA/0.1% Tween20. Membranes were washed 4 times with PBS/0.02% Tween 20. Proteins werevisualized with ECL Western Blotting Reagents (Amersham) according tothe manufacturer's instructions. Membranes were then exposed to XAR 5Scientific Imaging film (Kodak) for 5 seconds.

RESULTS

Rat monoclonal antibody RP57-16 reacts with both recombinant human andXenopus noggin and with recombinant human noggin produced in E. coli, ininsect cells, and in COS-M5 cells. The antibody does not react with theneurotrophins BDNF, NT-3 and NT-4. Western blotting showed that theantibody detects both reduced and non-reduced protein.

DEPOSIT OF MICROORGANISMS

The following were deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 under the terms of theBudapest Treaty:

    ______________________________________                                                       ATCC Accession                                                                          Date of                                                             No.       Deposit                                              ______________________________________                                        phage     hnogλ-5                                                                           75311       9-23-92                                      phage     hnogλ-7                                                                           75309       9-23-92                                      phage     hnogλ-9                                                                           75310       9-23-92                                      phage     hnogλ-10                                                                          75308       9-23-92                                      hybridoma RP57-16    CRL 11446   8-25-93                                      ______________________________________                                    

It is to be understood that while the invention has been described abovein conjunction with preferred specific embodiments, the description andexamples are intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 24                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 699 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..699                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGGAGCGCTGCCCCAGCCTAGGGGTCACCCTCTACGCCCTGGTGGTG48                            MetGluArgCysProSerLeuGlyValThrLeuTyrAlaLeuValVal                              151015                                                                        GTCCTGGGGCTGCGGGCGACACCGGCCGGCGGCCAGCACTATCTCCAC96                            ValLeuGlyLeuArgAlaThrProAlaGlyGlyGlnHisTyrLeuHis                              202530                                                                        ATCCGCCCGGCACCCAGCGACAACCTGCCCCTGGTGGACCTCATCGAA144                           IleArgProAlaProSerAspAsnLeuProLeuValAspLeuIleGlu                              354045                                                                        CACCCAGACCCTATCTTTGACCCCAAGGAAAAGGATCTGAACGAGACG192                           HisProAspProIlePheAspProLysGluLysAspLeuAsnGluThr                              505560                                                                        CTGCTGCGCTCGCTGCTCGGGGGCCACTACGACCCAGGCTTCATGGCC240                           LeuLeuArgSerLeuLeuGlyGlyHisTyrAspProGlyPheMetAla                              65707580                                                                      ACCTCGCCCCCCGAGGACCGGCCCGGCGGGGGCGGGGGTGCAGCTGGG288                           ThrSerProProGluAspArgProGlyGlyGlyGlyGlyAlaAlaGly                              859095                                                                        GGCGCGGAGGACCTGGCGGAGCTGGACCAGCTGCTGCGGCAGCGGCCG336                           GlyAlaGluAspLeuAlaGluLeuAspGlnLeuLeuArgGlnArgPro                              100105110                                                                     TCGGGGGCCATGCCGAGCGAGATCAAAGGGCTAGAGTTCTCCGAGGGC384                           SerGlyAlaMetProSerGluIleLysGlyLeuGluPheSerGluGly                              115120125                                                                     TTGGCCCAGGGCAAGAAGCAGCGCCTAAGCAAGAAGCTGCGGAGGAAG432                           LeuAlaGlnGlyLysLysGlnArgLeuSerLysLysLeuArgArgLys                              130135140                                                                     TTACAGATGTGGCTGTGGTCGCAGACATTCTGCCCCGTGCTGTACGCG480                           LeuGlnMetTrpLeuTrpSerGlnThrPheCysProValLeuTyrAla                              145150155160                                                                  TGGAACGACCTGGGCAGCCGCTTTTGGCCGCGCTACGTGAAGGTGGGC528                           TrpAsnAspLeuGlySerArgPheTrpProArgTyrValLysValGly                              165170175                                                                     AGCTGCTTCAGTAAGCGCTCGTGCTCCGTGCCCGAGGGCATGGTGTGC576                           SerCysPheSerLysArgSerCysSerValProGluGlyMetValCys                              180185190                                                                     AAGCCGTCCAAGTCCGTGCACCTCACGGTGCTGCGGTGGCGCTGTCAG624                           LysProSerLysSerValHisLeuThrValLeuArgTrpArgCysGln                              195200205                                                                     CGGCGCGGGGGCCAGCGCTGCGGCTGGATTCCCATCCAGTACCCCATC672                           ArgArgGlyGlyGlnArgCysGlyTrpIleProIleGlnTyrProIle                              210215220                                                                     ATTTCCGAGTGCAAGTGCTCGTGCTAG699                                                IleSerGluCysLysCysSerCys                                                      225230                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 232 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGluArgCysProSerLeuGlyValThrLeuTyrAlaLeuValVal                              151015                                                                        ValLeuGlyLeuArgAlaThrProAlaGlyGlyGlnHisTyrLeuHis                              202530                                                                        IleArgProAlaProSerAspAsnLeuProLeuValAspLeuIleGlu                              354045                                                                        HisProAspProIlePheAspProLysGluLysAspLeuAsnGluThr                              505560                                                                        LeuLeuArgSerLeuLeuGlyGlyHisTyrAspProGlyPheMetAla                              65707580                                                                      ThrSerProProGluAspArgProGlyGlyGlyGlyGlyAlaAlaGly                              859095                                                                        GlyAlaGluAspLeuAlaGluLeuAspGlnLeuLeuArgGlnArgPro                              100105110                                                                     SerGlyAlaMetProSerGluIleLysGlyLeuGluPheSerGluGly                              115120125                                                                     LeuAlaGlnGlyLysLysGlnArgLeuSerLysLysLeuArgArgLys                              130135140                                                                     LeuGlnMetTrpLeuTrpSerGlnThrPheCysProValLeuTyrAla                              145150155160                                                                  TrpAsnAspLeuGlySerArgPheTrpProArgTyrValLysValGly                              165170175                                                                     SerCysPheSerLysArgSerCysSerValProGluGlyMetValCys                              180185190                                                                     LysProSerLysSerValHisLeuThrValLeuArgTrpArgCysGln                              195200205                                                                     ArgArgGlyGlyGlnArgCysGlyTrpIleProIleGlnTyrProIle                              210215220                                                                     IleSerGluCysLysCysSerCys                                                      225230                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlnMetTrpLeuTrpSerGlnThrPheCysProValLeuTyr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ArgPheTrpProArgTyrValLysValGlySerCys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       SerLysArgSerCysSerValProGluGlyMetValCysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuArgTrpArgCysGlnArgArg                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       IleSerGluCysLysCysSerCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1834 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 595..1263                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CTAATAAATCCTAAGTAGCCAGAGGGACGAGCTACAGACTGGTTGCGGCGCAGGGTTTAT60                CCAGGGCAGAGAGGAGCAGCAAAAGCACATTGCGCAGCTCTCACTCCCCCTTTCCTTCTG120               CTTCACTCTATAAGGGCTCCTGCAAATGAAAGAGACCTGCGGGGATTTGCGCGGACAGAT180               GTAAAGGAGATCCTGCAACTTTCTCTGGTTGCATCCCTGGGAGTCGCTGCGCGCCGCTGG240               CTGATTGCGACTGTTGCTTTCCACAGCTCCCTTCTTCCGCAGTTTCTTCTAGGAGCAGAT300               CGAGTCTCTGGTTACCATGGTGATCGAGCTGAAAGTGAAGAATATTTAAGAGAGGGGAGG360               CTGGAGCCAGCAGGCAGACAAAGTGGTGCCACCAAGGACTGTGCGTAAAGGGTGAGCGCA420               TTGGAGACAGACAGGGGCTCTGCTGAACTTCCACTTGACTGCGATGAGAGGGGGGAATCC480               CCAATTCGCTAGGTGCCCCTGAACCCCCCAGAATTCCTCCTCTGATGCATTATTTATGAT540               CTCTGGCAAGAAATCGGGAGCACCCAACTCTTATTTTGTGCAGCTGTGTGCAGCATG597                  Met                                                                           GATCATTCCCAGTGCCTTGTGACTATATATGCTCTGATGGTCTTCTTG645                           AspHisSerGlnCysLeuValThrIleTyrAlaLeuMetValPheLeu                              51015                                                                         GGACTTAGAATAGACCAAGGGGGTTGCCAACATTATCTGCACATCAGA693                           GlyLeuArgIleAspGlnGlyGlyCysGlnHisTyrLeuHisIleArg                              202530                                                                        CCGGCTCCTAGTGAAAACCTACCACTGGTGGACCTTATTGAGCACCCG741                           ProAlaProSerGluAsnLeuProLeuValAspLeuIleGluHisPro                              354045                                                                        GATCCCATCTATGATCCCAAGGAGAAGGATCTTAACGAGACCTTGCTG789                           AspProIleTyrAspProLysGluLysAspLeuAsnGluThrLeuLeu                              50556065                                                                      AGGACTTTAATGGTTGGACACTTTGACCCCAACTTTATGGCCACCATC837                           ArgThrLeuMetValGlyHisPheAspProAsnPheMetAlaThrIle                              707580                                                                        CTGCCAGAGGAGAGACTTGGAGTGGAGGACCTTGGGGAGTTGGATCTC885                           LeuProGluGluArgLeuGlyValGluAspLeuGlyGluLeuAspLeu                              859095                                                                        CTTCTTAGGCAGAAGCCCTCGGGGGCAATGCCAGCGGAAATCAAGGGA933                           LeuLeuArgGlnLysProSerGlyAlaMetProAlaGluIleLysGly                              100105110                                                                     CTGGAGTTTTACGAGGGGCTTCAGAGCAAAAAGCACAGACTGAGCAAG981                           LeuGluPheTyrGluGlyLeuGlnSerLysLysHisArgLeuSerLys                              115120125                                                                     AAACTCAGGAGAAAGTTGCAGATGTGGCTCTGGTCCCAGACCTTCTGT1029                          LysLeuArgArgLysLeuGlnMetTrpLeuTrpSerGlnThrPheCys                              130135140145                                                                  CCTGTCCTTTACACATGGAATGACCTAGGGACCAGGTTTTGGCCTCGC1077                          ProValLeuTyrThrTrpAsnAspLeuGlyThrArgPheTrpProArg                              150155160                                                                     TATGTGAAAGTAGGGAGCTGCTACAGTAAGAGGTCTTGTTCTGTGCCA1125                          TyrValLysValGlySerCysTyrSerLysArgSerCysSerValPro                              165170175                                                                     GAGGGCATGGTTTGCAAAGCTGCCAAGTCTATGCATTTGACCATCTTA1173                          GluGlyMetValCysLysAlaAlaLysSerMetHisLeuThrIleLeu                              180185190                                                                     AGGTGGAGATGTCAACGCAGGGTTCAGCAGAAGTGTGCGTGGATAACC1221                          ArgTrpArgCysGlnArgArgValGlnGlnLysCysAlaTrpIleThr                              195200205                                                                     ATTCAGTACCCTGTCATTTCCGAGTGCAAATGCTCATGCTGAGACTCTT1270                         IleGlnTyrProValIleSerGluCysLysCysSerCys                                       210215220                                                                     GGACTAATGCAAAAAGACAGTAGCTTCATGGTTCAAGCTTCATGTTATATGCACTGTAAT1330              ATGTAGAAATGTATATGTGTGTATATATGGCATTGGTCTAAATTACTATTAAAAGGTCAG1390              TATTATTCTTTTAAATAACCAGTGTCTACTGTATTTCCAACACTATTATCCTGGTTGTGT1450              TTTATTTTAATAATATTATTATTATTTTTTTTTTGCCTAATGTATCTCTATTTATATCCA1510              AAAAAAGAGCACTTCGCTTGGCGAAGCATTTTTTTTTAAAGAAAAAAAAAACAAATTTAA1570              TAGTTTAATAATATAGAAGCATTTTTTTCCTTTAATGGAAAATGTGCCTTTTTTTGATGG1630              ACCTCAAAAAAAAAATGAATAAAAACCAGAGCAAGATATAATTTTCAGTTTATTGTACAT1690              AGAAAGAGAACACATTTTGAAAGATGAAAAATTTTACTCTTGTAAATGAGAACTATCTGC1750              TATTTATTCTTTTATTTTTTTTTTCCTCCCCCTGTAGAGTGCAGAATAAAAGTAAACCAC1810              TAAAATATTAAAAAAAAAAAAAAA1834                                                  (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 222 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetAspHisSerGlnCysLeuValThrIleTyrAlaLeuMetValPhe                              151015                                                                        LeuGlyLeuArgIleAspGlnGlyGlyCysGlnHisTyrLeuHisIle                              202530                                                                        ArgProAlaProSerGluAsnLeuProLeuValAspLeuIleGluHis                              354045                                                                        ProAspProIleTyrAspProLysGluLysAspLeuAsnGluThrLeu                              505560                                                                        LeuArgThrLeuMetValGlyHisPheAspProAsnPheMetAlaThr                              65707580                                                                      IleLeuProGluGluArgLeuGlyValGluAspLeuGlyGluLeuAsp                              859095                                                                        LeuLeuLeuArgGlnLysProSerGlyAlaMetProAlaGluIleLys                              100105110                                                                     GlyLeuGluPheTyrGluGlyLeuGlnSerLysLysHisArgLeuSer                              115120125                                                                     LysLysLeuArgArgLysLeuGlnMetTrpLeuTrpSerGlnThrPhe                              130135140                                                                     CysProValLeuTyrThrTrpAsnAspLeuGlyThrArgPheTrpPro                              145150155160                                                                  ArgTyrValLysValGlySerCysTyrSerLysArgSerCysSerVal                              165170175                                                                     ProGluGlyMetValCysLysAlaAlaLysSerMetHisLeuThrIle                              180185190                                                                     LeuArgTrpArgCysGlnArgArgValGlnGlnLysCysAlaTrpIle                              195200205                                                                     ThrIleGlnTyrProValIleSerGluCysLysCysSerCys                                    210215220                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 356 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..265                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 320                                                             (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCAGATGTGGCTGTGGTCACAGACCTTCTGCCCGGTGCTGTACGCG46                              GlnMetTrpLeuTrpSerGlnThrPheCysProValLeuTyrAla                                 151015                                                                        TGGAATGACCTAGGCAGCCGCTTTTGGCCACGCTACGTGAAGGTGGGC94                            TrpAsnAspLeuGlySerArgPheTrpProArgTyrValLysValGly                              202530                                                                        AGCTGCTTCAGCAAGCGCTCCTGCTCTGTGCCCGAGGGCATGGTGTGT142                           SerCysPheSerLysArgSerCysSerValProGluGlyMetValCys                              354045                                                                        AAGCCATCCAAGTCTGTCAGCCTCACGGTGCTGCGGTGGCGCTGTCAG190                           LysProSerLysSerValSerLeuThrValLeuArgTrpArgCysGln                              505560                                                                        CGGCGCGGGGGTCAGCGCTGCGGCTGGATTCCCATCCAGTACCCCATC238                           ArgArgGlyGlyGlnArgCysGlyTrpIleProIleGlnTyrProIle                              657075                                                                        ATTTCCGAGTGTAAGTGTTCCTGCTAGAACTCGGGGGGGCCCCCTGCCCGCGCC292                     IleSerGluCysLysCysSerCys                                                      8085                                                                          CAGACACTTGATGGATCCCCACCAACGNCCCCCCTACCCCCACCACCTCCAACCAGTTTC352               ACCA356                                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlnMetTrpLeuTrpSerGlnThrPheCysProValLeuTyrAlaTrp                              151015                                                                        AsnAspLeuGlySerArgPheTrpProArgTyrValLysValGlySer                              202530                                                                        CysPheSerLysArgSerCysSerValProGluGlyMetValCysLys                              354045                                                                        ProSerLysSerValSerLeuThrValLeuArgTrpArgCysGlnArg                              505560                                                                        ArgGlyGlyGlnArgCysGlyTrpIleProIleGlnTyrProIleIle                              65707580                                                                      SerGluCysLysCysSerCys                                                         85                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 15                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CARACNTTYTGYCCNGTN18                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= M                                                                     /note= "M = A or C."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 15                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 21                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 24                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TTYTGGCCNMGNTAYGTNAARGTNGG26                                                  (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 15                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCNGARGGNATGGTNTG17                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= S                                                                     /note= "S = C or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= W                                                                     /note= "W = A or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 15                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CANSWRCAYTTRCAYTC17                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 15                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CANACCATNCCYTCNGG17                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= K                                                                     /note= "K = G or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= K                                                                     /note= "K = G or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= N                                                                     /note= "N = A or C or G or T."                                                (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 14                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= K                                                                     /note= "K = G or T."                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CKNCKYTGRCANCKCCA17                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..18                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CAGATGTGGCTGTGGTCA18                                                          GlnMetTrpLeuTrpSer                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GlnMetTrpLeuTrpSer                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GCAGGAACACTTACACTC18                                                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GluCysLysCysSerCys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /mod.sub.-- base=i                                     /label= N                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod.sub.-- base=i                                     /label= N                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 15                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= Y                                                                     /note= "Y = C or T."                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /mod.sub.-- base=OTHER                                 /label= R                                                                     /note= "R = A or G."                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GARGGNATGGTNTGYAARCC20                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GACTCGAGTCGACATCGCAGATGTGGCTGTGGTCAC36                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CCAAGCTTCTAGAATTCGCAGGAACACTTACACTCGG37                                       __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule having a nucleotidesequence selected from the group consisting of:(a) a nucleotide sequencecomprising the coding region of human noggin contained in the vectordesignated as hnogX-9 (ATCC Accession No. 75310); and (b) a nucleotidesequence differing from the sequence of (a) and which encodes apolypeptide encoded by the sequence of (a).
 2. A vector which comprisesthe isolated nucleic acid molecule of claim
 1. 3. An expression vectorcomprising the nucleic acid molecule of claim 1, wherein the molecule isoperatively linked to an expression control sequence.
 4. A host-vectorsystem for the production of a polypeptide having the functionalactivity of human noggin which comprises the expression vector of claim3, in a cultured host cell.
 5. The host-vector system of claim 4,wherein the host cell is a bacterial cell.
 6. The host-vector system ofclaim 5, wherein the bacterial cell is E. coli.
 7. The host-vectorsystem of claim 4, wherein the host cell is a yeast cell.
 8. Thehost-vector system of claim 4, wherein the host cell is an insect cell.9. The host-vector system of claim 4, wherein the host cell is amammalian cell.
 10. An isolated nucleic acid molecule having anucleotide sequence selected from the group consisting of:(a) anucleotide sequence comprising the coding region of human noggin as setforth in SEQ ID No.1; and (b) a nucleotide sequence differing from thesequence of (a) and which encodes a polypeptide encoded by the sequenceof (a).
 11. A vector which comprises the isolated nucleic acid moleculeof claim
 10. 12. An expression vector comprising the nucleic acidmolecule of claim 10, wherein the molecule is operatively linked to anexpression control sequence.
 13. A host-vector system for the productionof a polypeptide having the functional activity of human noggin whichcomprises the expression vector of claim 12, in a cultured host cell.14. The host-vector system of claim 13, wherein the host cell is abacterial cell.
 15. The host-vector system of claim 14, wherein thebacterial cell is E. coli.
 16. The host-vector system of claim 13,wherein the host cell is a yeast cell.
 17. The host-vector system ofclaim 13, wherein the host cell is an insect cell.
 18. The host-vectorsystem of claim 13, wherein the host cell is a mammalian cell.
 19. Anisolated nucleic acid molecule encoding human noggin having the aminoacid sequence as depicted in SEQ ID No.2.
 20. Isolated human nogginencoded by a nucleotide sequence comprising the nucleotide sequence asset forth in SEQ. ID. NO.1.
 21. A composition comprising the isolatedhuman noggin of claim 20, and a vehicle.
 22. Isolated human noggincomprising the amino acid sequence as set forth in SEQ ID No.2.
 23. Acomposition comprising the isolated human noggin of claim 22, and avehicle.